Pasteurella multocida toxin derivatives

ABSTRACT

A vaccine for immunizing animals against diseases caused by microorganisms producing an osteolytic toxin comprising an immunologically effective amount of a recombinant, immunogenic, detoxified Pasteurella multocida toxin or toxin analog, the vaccine being encoded by a nucleotide sequence coding for a Pasteurella multocida toxin or toxin analog which is inserted in an expression vector capable of replicating in a suitable host microorganism in which the said sequence may be expressed. Optionally, the produced toxin or toxin analog is subjected to posttranscriptional modifications to provide the detoxified toxin or toxin analog. Alternatively, the nucleotide sequence may be modified prior to insertion into the expression vector so as to provide the toxin or toxin analog in a detoxified form. Furthermore, the use of a monoclonal antibody against Pasteurella multocida toxin or toxin analog, a labelled DNA sequence homologous to a DNA sequence coding for a toxin or toxin analog, and the toxin or toxin analog per se as diagnostic agents are described.

This application is a continuation of application Ser. No. 07/582,945,filed Oct. 12, 1990 which is the national stage of PCT/DK89/00084 filedApr. 11, 1989.

FIELD OF INVENTION

The present invention relates to a vaccine for immunizing animalsagainst diseases caused by microorganisms producing an osteolytic toxin,a DNA sequence encoding a Pasteurella multocida toxin useful forproducing the toxin and as a diagnostic agent, methods of producing andisolating a P. multocida toxin, use of a P. multocida toxin, amonoclonal antibody directed against a P. multocida toxin, a diagnosticagent comprising said monoclonal antibody and the use of said monoclonalantibody for a variety of diagnostic and other purposes.

TECHNICAL BACKGROUND

Atrophic rhinitis is a disease which profoundly affects the bonestructure of, the porcine snout. The etiological agent which iscurrently considered to be the cause of growth retarding progressiveatrophic rhinitis is toxigenic (toxin-producing) strains of P. multocidawhich colonize the nasal cavity of pigs (Pedersen and Barfod, 1981,(ref. 1), Rutter and Rojas, 1982, (ref. 2), Elling and Pedersen, 1985,(ref. 3), Pedersen et al. 1988 (ref. 4). It has been shown that thenasal mucosa are more easily colonized by P. multocida when theresistance to infection is lower such as when the pigs are concomitantlyinfected with Bordetella bronchiseptica or when the nasal mucosa areexposed to a mild chemical irritant (cf. Pedersen and Elling, 1984,(ref. 5).

The pathological effects of P. multocida infection may be ascribed to atoxin produced by this bacterium. The toxin which has an apparentmolecular weight of 143 kd and an actual molecular weight of 146.5 kdinduces bone resorption (osteolysis) of the nasal turbinates and otherbone structures in the nasal cavity by stimulating osteoclast activityin porcine turbinate bones, and causes impaired osteoblastic boneformation.

The disease is of major economic importance to pig breeders all over theworld, since apart from the pathological effects on the nasal (andoccasionally facial) bones noted above, it causes a slower growth rateof the infected pigs and consequently higher production costs. Attemptshave therefore been made to reduce the occurrence and the significanceof P. multocida infection, for instance by the establishment of SPF(specific pathogen free) pigs via cesarean section, or by antibiotictreatment of infected animals or prophylactic vaccination.

Known vaccines for the immunization of animals, principally pigs,against diseases ascribable to P. multocida infection, especiallyatrophic rhinitis, comprise killed P. multocida cells optionallycombined with killed Bordetella bronchiseptica cells (cf. EP 85 469)and/or an inactivated (usually by heat treatment or addition offormaldehyde) toxin-containing extract of toxigenic P. multocida.Vaccines of the latter type are commercially available from NorthernDrugs & Chemicals Ltd., Copenhagen, Denmark, under the trademarkAtrinord®, as well as from Intervet International BV, Boxmeer, Holland,under the trademark Nobi-vacART®.

The present inventors contemplate that an improved immunogenic effectrelative to the known vaccine preparations may be obtained by using apurified and suitably modified toxin preparation for vaccinationpurposes either to replace the conventional vaccines or as a constituentthereof.

The purification of P. multocida toxin has previously been described.Thus, Foged et al., 1987, (ref. 6) disclose the purification of thetoxin by chromatography and polyacrylamide gel electrophoresis. Thepurified toxin is used solely for studying its toxic and pathologicaleffects. Kamp et al., 1987, (ref. 7) also disclose the purification ofthe P. multocida toxin for the purpose of clinical studies. They suggestthat the purified toxin may be used as an antigen to raise specificantibodies useful for serological tests. Nakai et al., 1984, (ref. 8)disclose a method of purifying the P. multocida toxin by chromatographyand polyacrylamide gel electrophoresis. They further disclose theproduction of polyclonal antibodies directed against the purified toxinwhich they use to analyse the purity of the purified toxin. It issuggested that the antibodies may be used to further study the role ofthe toxin in atrophic rhinitis.

None of these publications suggest the use of a purified toxin as acomponent of a vaccine for immunizing animals against Pasteurellainfection, and this is believed to be a novel concept.

SUMMARY OF THE INVENTION

Accordingly, in one aspect the present invention relates to a vaccinefor immunizing an animal, including a human being, against diseasescaused by microorganisms producing an osteolytic toxin, the vaccinecomprising an immunogenically effective amount of a recombinant,immunogenic, detoxified P. multocida toxin or toxin analogue togetherwith an immunologically acceptable carrier or vehicle.

For the preparation of the known vaccines, a toxigenic Pasteurellastrain is cultivated and the toxin is isolated from the culture mediumor from a bacterial extract followed by detoxification by, for instance,thermal or chemical treatment. Compared to this procedure, theproduction of the toxin or toxin analogue by recombinant DNA techniqueshas a number of advantages: it is possible to produce the toxin or toxinanalogue by growing a non-pathogenic organism, the toxin or toxinanalogue may be produced in higher quantities than those produced bywild-type P. multocida strains, for instance by using a strong promoterto induce a high level of expression of the toxin gene or by using ahigh copy number vector for cloning the toxin gene, and it is possibleto produce the toxin or toxin analogue in a detoxified form, e.g. bysubjecting the gene encoding the toxin to treatment with a mutagen, orby deleting a part of the nucleotide sequence coding for the toxin ortoxin analogue, substituting one or more nucleotides in the sequence,etc. The recombinant toxin or toxin analogue may be used insubstantially pure form in the vaccine of the invention but may also beemployed as a crude or partially purified preparation.

In the present context, the term "substantially pure" is understood tomean that the vaccine is substantially free from other immunogenicallyactive components the presence of which might give rise to adverseimmune reactions in the animals immunized with the vaccine and, mostimportantly, that no other components of the microorganisms producingthe toxin or toxin analogue, such as cell debris or cellular proteinsapart from the toxin or toxin analogue itself or a protein orpolypeptide to which the toxin or toxin analogue is fused (vide below)are present in the vaccine preparation. A high purity of the detoxifiedtoxin or toxin analogue is believed to result in a high antitoxinresponse on immunization with the vaccine of the invention and a lowerdosage of the toxin or toxin analogue may consequently be required forimmunization purposes than that used in crude or partially purifiedvaccine preparations. A substantially pure toxin or toxin analogue hasthe added advantage that the exact concentration thereof in a givenvaccine preparation is known so that an exact dosage may be administeredto the animal in question.

The microorganism producing an osteolytic toxin (i.e. a toxin directlyor indirectly involved in bone resorption) against which the vaccineconfers immunity is preferably P. multocida. Other microorganisms whichhave shown osteolytic effects or regulation of specific markers of bonemetabolism are e.g. Actinomyces viscosus and Bordetella pertussis(Trummel et al., 1979, (ref. 9) and Price (ref. 10).

Due to the toxic activity of the P. multocida toxin, it is not possibleto use the native toxin in a vaccine of the invention. Rather, it mustbe present in detoxified form. The term "detoxified" should beunderstood to mean that the toxic activity has been removed from atleast a sufficient number, but not necessarily all, of the toxinmolecules present in the vaccine preparation so that the vaccine, whenadministered to an animal to be immunized, will not produce any adversetoxic effects in the animal in question, while still giving rise to asatisfactory immune response.

The detoxification of the P. multocida toxin or toxin analogue may becarried out in a variety of ways. Thus, it is possible to subject thetoxin or toxin analogue to thermal treatment, the toxin being known tobe heat labile and to be inactivated (i.e. detoxified) at 70° C.Furthermore, the toxin or toxin analogue may be subjected to treatmentwith a chemical, such as formaldehyde, glutaraldehyde or a suitableproteolytic enzyme, e.g. trypsin. Detoxification may also be broughtabout by mutagenizing the gene coding for the P. multocida toxin ortoxin analogue by means of, for instance, ultraviolet radiation,ionizing radiation or a chemical mutagen such as mitomycin C,5-bromouracil, methylmethane sulphonate, nitrogen mustard or anitrofuran. Furthermore, the toxin may be detoxified by substitution,deletion, addition or insertion of one or more amino acids in the toxinor toxin analogue, or by substitution, addition, deletion or insertionof one or more base pairs in the nucleotide sequence coding for thetoxin or toxin analogue, or a combination of these measures.

In contrast to detoxification by thermal or chemical treatment, thegenetic procedure has the obvious advantage of resulting in a uniformpopulation of equally detoxified molecules.

It should be noted that the terms "substitution, deletion, addition orinsertion" should be interpreted with reference to the full-length toxinprotein. Thus, "substitution" is intended to mean the replacement of anyone or more amino acids or nucleotides in the full amino acid ornucleotide sequence with one or more others, "addition" is understood tomean the addition of one or more amino acids or nucleotides at eitherend of the full amino acid or nucleotide sequence, "insertion" isintended to mean the introduction of one or more amino acids ornucleotides within the full amino acid or nucleotide sequence, and"deletion" is intended to indicate that one or more amino acids ornucleotides have been deleted from the full amino acid or nucleotidesequence whether at either end of the sequence or at any suitable pointwithin it. It should be understood that the detoxification of the toxinor toxin analogue may also be brought about by a combination of two ormore of these procedures.

The term "toxin analogue" is used in the present context to indicate aprotein or polypeptide of a similar amino acid composition or sequenceas the P. multocida toxin, allowing for variations which do not have anadverse effect on the immunogenicity of the analogue.

The analogous polypeptide or protein may be derived from a microorganismof another species than P. multocida or may be partially or completelyof synthetic origin. The analogous polypeptide or protein may also beone which comprises at least one epitope reactive with anti-P. multocidatoxin antibodies found in samples from individuals with atrophicrhinitis and/or which elicits antibodies reactive with native P.multocida toxin. The term is further intended to mean any immunogenicsubsequence, functional equivalent or derivative of the toxin.

The term "immunogenic subsequence" is intended to indicate a sequence ofthe full-length toxin which from the outset is produced in a truncatedform relative to the full-length toxin protein or which subsequent toproduction of the full-length protein is generated, for instance byproteolytic cleavage thereof or by expression of a nucleotide sequenceshorter than the full nucleotide sequence encoding P. multocida toxin.The minimum subsequence is one which at least comprises a relevantepitope of the toxin, i.e. an epitope which gives rise to a relevantimmune response in an animal immunized with the vaccine of theinvention.

The term "functional equivalent" is intended to include allimmunogenically active substances with the ability to evoke an immuneresponse in animals to which a vaccine containing the equivalent hasbeen administered which is similar to the immune response evoked by thedetoxified P. multocida toxin, in that it is able to confer immunity todiseases caused by microorganisms producing an osteolytic toxin. Thefunctional equivalent may be derived from a microorganism of anotherspecies than P. multocida or may partially or completely be of syntheticorigin. It should be understood that the similarities between the P.multocida toxin and the functional equivalent are qualitative ratherthan quantitative, relating to the nature rather than the level ofactivity of the functional equivalent.

The term "derivative" is understood to mean a modification of the toxinsuch as one produced by substitution, insertion, addition or deletion ofone or more amino acids or nucleotides or a combination of thesemeasures, as defined above, or by fusion with another polypeptide.

In a further aspect, the present invention relates to a DNA fragmentcomprising a nucleotide sequence coding for a P. multocida toxin ortoxin analogue, as defined above. The DNA fragment may for instance beused in a method of preparing the toxin or toxin analogue by recombinantDNA techniques or as a diagnostic agent (i.e. a DNA probe).

In a still further aspect, the present invention relates to a monoclonalantibody which is directed against or reactive with a P. multocida toxinor a toxin analogue as defined above, or a fragment of said antibody. Itshould be noted that the antibody may be reactive with both the toxicand detoxified toxin, thus making it useful for a variety of diagnostic,immunization and isolation purposes as will be described in furtherdetail below.

DETAILED DISCLOSURE OF THE INVENTION

The toxin produced by P. multocida (in the following occasionallyabbreviated to PMT) which, as noted above, is generally believed to bethe causative agent of porcine atrophic rhinitis, has in the priorliterature been variously termed "dermonecrotic toxin", "osteolytictoxin", "turbinate atrophy toxin" and "heat labile exotoxin", but itwould appear to be the same toxin as the amino acid composition,isoelectric point and biological activities of the variously termedtoxins show basic similarities, although minor variations in theproperties of toxins isolated from different strains of P. multocidaappear to exist. The estimated amino acid composition of PMT (as deducedfrom the DNA sequence) is as follows:

    ______________________________________                                                 Ala is found 76 times = 5.91%                                                 Cys is found 8 times = 0.62%                                                  Asp is found 71 times = 5.53%                                                 Glu is found 100 times = 7.78%                                                Phe is found 69 times = 5.37%                                                 Gly is found 71 times = 5.53%                                                 His is found 19 times = 1.48%                                                 Ile is found 92 times = 7.16%                                                 Lys is found 70 times = 5.45%                                                 Leu is found 127 times = 9.88%                                                Met is found 36 times = 2.80%                                                 Asn is found 73 times = 5.68%                                                 Pro is found 62 times = 4.82%                                                 Gln is found 56 times = 4.36%                                                 Arg is found 58 times = 4.51%                                                 Ser is found 97 times = 7.55%                                                 Thr is found 66 times = 5.14%                                                 Val is found 63 times = 4.90%                                                 Trp is found 18 times = 1.40%                                                 Tyr is found 53 times = 4.12%                                        ______________________________________                                    

The total number of amino acid residues is 1285, and the full-lengthtoxin has a molecular weight of 146.5 kd.

The recombinant toxin or toxin analogue used in the vaccine of theinvention may more specifically be one encoded by a DNA sequencesubstantially as shown in FIGS. 10 (a)-(j) (SEQ ID NO:1) or asubsequence thereof encoding an immunogenic subsequence of the toxin ortoxin analogue. It should be noted that the amino acid sequence deducedfrom the DNA sequence is also shown in FIGS. 10 (a)-(j) (SEQ ID NO:2)above the DNA sequence. A suitable analogue may be one which has a DNAsequence which differs from that of the native toxin by one or more basepairs and which may be derived by substituting one or more nucleotidesin the toxin DNA sequence either giving rise to the same amino acidsequence, but where the nucleotide substitutions make the sequenceconform to the codon usage of the microorganism in which the sequence isinserted, or giving rise to a somewhat different amino acid sequencewhich, however, is functionally similar to that of the native toxin.

Apart from the toxin or toxin analogue as defined above, the vaccine ofthe invention also comprises an immunologically acceptable carrier orvehicle. This vehicle may be any vehicle usually employed in thepreparation of vaccines, e.g. a diluent such as isotonic saline,suspending agent etc. The vaccine may be prepared by mixing animmunogenically effective amount of the toxin or toxin analogue with thevehicle in an amount resulting in the desired concentration of the toxinor toxin analogue in the vaccine preparation. Although the amount oftoxin or toxin analogue per unit dose of the vaccine will differaccording to the age of the animals to be immunized (for instanceaccording to whether sows or piglets are to be immunized against P.multocida), the route and form of administration, and the immunogenicityof the particular toxin present in the vaccine, a suitable amount oftoxin or toxin analogue is contemplated in the range of 0.1-500 μg perdosage of the vaccine.

The vaccine may further comprise an adjuvant in order to increase theimmunogenicity of the vaccine preparation. The adjuvant may be selectedfrom Freund's complete or incomplete adjuvant, aluminium hydroxide,Bordetella pertussis, a saponin, a muramyl dipeptide, an iscom (immunestimulating complex; cf. for instance EP 109 942) and an oil, such as avegetable oil, e.g. peanut oil, or a mineral oil, e.g. silicone oil.

In some cases it may be advantageous to couple the toxin or toxinanalogue to a carrier, in particular a macromolecular carrier. Thecarrier is usually a polymer to which the toxin is bound by hydrophobicnon-covalent interaction, such as a plastic, e.g. polystyrene, or apolymer to which the toxin is covalently bound, such as apolysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbuminor keyhole limpet hemocyanin. The carrier should preferably be non-toxicand non-allergenic. The toxin or toxin analogue may be multivalentlycoupled to the macromolecular carrier as this provides an increasedimmunogenicity of the vaccine preparation. It is also contemplated thatthe toxin or toxin analogue may be presented in multivalent form bypolymerizing the toxin or toxin analogue with itself.

In a particular embodiment of the vaccine of the present invention, thetoxin or toxin analogue as defined above is fused to anotherpolypeptide. Techniques for preparing fused polypeptides are known from,e.g. Casadaban and Cohen, 1983, (ref. 11). Alternatively, the fusion maybe provided by fusing the nucleotide sequence encoding the toxin to anucleotide sequence encoding another polypeptide so that the fusednucleotide sequence, when inserted in an appropriate vector, isexpressed as a fusion polypeptide on transformation of the vector to asuitable microorganism and growth of the microorganism under conditionsfavorable to the expression of the fused sequence. The polypeptide towhich the toxin is fused may, for instance, be a carrier polypeptide assuggested above, lysozyme or another immunogenic peptide such as a Typrotein from Saccharomyces cerevisiae, protein A from Staphylococcusaureus, Hepatitis B core antigen, etc.

It is also contemplated that the vaccine may be in the form of tablet,granule or capsule intended for oral administration since there is someevidence that immunogens may be absorbed through the intestinal wall andstimulate B-lymphocytes which then migrate to local epithelial regionswhere they transform into immunoglobulin-secreting plasma cells. An oralvaccine should be provided with an enteric coating in order to protectthe toxin or toxin analogue from substances present in gastric juicewhich might be deleterious to the toxin or toxin analogue, such aspepsin. The enteric coating may be selected from shellac, celluloseacetate esters such as cellulose acetate phthalate, hydroxypropylmethylcellulose esters such as hydroxypropylmethyl cellulose phthalate,polyvinyl acetate esters such as polyvinyl acetate phthalate, andpolymers of methacrylic acid and (meth)acrylic acid esters. Newlydeveloped methods of encapsulations, based on microspheres with adiameter of about 5-15 μm are of special interest since such particlescontaining an immunogenic substance after administration will beselectively delivered to Peyers's patches thereby providing immunity onmucosal surfaces. Stimulation of the immune response on respiratorymucosal surfaces may also be obtained through intranasal immunizations.(Mestecky, 1987, (ref. 12).

The DNA molecule or DNA fragment of the invention comprising thenucleotide sequence encoding the toxin or toxin analogue may be derivedfrom complementary cDNA obtained by preparing a cDNA library on thebasis of mRNA from a toxin-producing P. multocida strain by standardmethods. Alternatively and preferably, the nucleotide sequence may bederived from a P. multocida genome, by screening for genomic sequenceshybridizing to a DNA probe prepared on the basis of the full or partialamino acid sequence of the toxin in accordance with establishedprocedures or by establishing a toxin gene library and screening fortoxin-producing clones by means of a toxin-specific antibody (for a moredetailed description of this procedure, see Example 4). In the case ofPMT, it is not possible to prepare a DNA probe on the basis of itsN-terminal amino acid sequence since PMT is blocked in the N-terminaland therefore is not degraded by procedures for the sequencing of aminoacids.

Another routine screening method which has proven to be difficult in thecase of PMT is screening for toxin-producing clones by means of ananti-PMT serum. When using serum from a rabbit repeatedly immunized withPMT, the present inventors found 5 E. coli clones by the Colony blotmethod in the gene library described in Example 5. Further studies ofthe above 5 clones, however, showed that none of them were producingPMT. These results indicate the importance of performing the screeningwith anti-PMT monoclonal antibodies as described in Example 5.

The nucleotide sequence may also be derived from a bacteriophageinfectious for P. multocida, i.e. one which has been transferred fromone bacterial strain which originally carried the sequence to anotherstrain which did not originally carry the sequence by bacteriophagetransfection. Similarly, the nucleotide sequence may be derived from aplasmid or other genetic element transferred from one strain to anotherby conjugation, transformation or the like.

Furthermore, the nucleotide sequence coding for the toxin may be asynthetic sequence, that is, one prepared according to standardprocedures, e.g. as described in Matthes et al., 1984, (ref. 13).Finally, the nucleotide sequence may be a mixed genomic and synthetic ormixed cDNA and synthetic sequence prepared by ligating DNA fragments ofgenomic, cDNA or synthetic origin (as appropriate) which DNA fragmentseach contain part of the nucleotide sequence encoding the toxin, inaccordance with established methods.

In accordance with the explanation given above, the DNA molecule or DNAfragment may be one which has been modified by substitution, addition,insertion or deletion of one or more nucleotides in the sequence withthe purpose of establishing a sequence which, when expressed, results inthe production of a detoxified toxin or toxin analogue.

In particular, the invention relates to a DNA fragment which comprises anucleotide sequence substantially as shown in FIGS. 10 (a)-(j) (SEQ IDNO:1) or a modification thereof as indicated above. The sequence codingfor the full-length toxin starts at position 219 (or 213) of thesequence shown in the figure, while the end of the sequence is atposition 4073. The DNA sequence shown in FIGS. 10 (a)-(j) (SEQ ID NO:1)has been established by well-known methods as described in Example 7below.

The DNA molecule or DNA fragment of the invention may further comprise anucleotide sequence encoding another polypeptide fused to the nucleotidesequence encoding the toxin or toxin analogue with the purpose ofproducing a fused polypeptide, as explained above. A further purpose ofpreparing a fused polypeptide may be to facilitate purification of thetoxin. In this case, the fused sequence may be inserted into anappropriate vector which is transformed to a suitable host microorganismwhich is grown under conditions ensuring expression of the fusedsequence after which the fused polypeptide is recovered from the cultureby subjecting the fused polypeptide to affinity chromatography involvingan antibody or any other ligand reacting with the second polypeptide.After purification, the second polypeptide may then be removed, forinstance by suitable proteolytic cleavage followed by separation of thetwo polypeptides.

In a further aspect, the invention relates to an expression vector whichis capable of replicating in a host microorganism and which carries aDNA molecule or DNA fragment as described above. The vector may eitherbe one which is capable of autonomous replication, such as a plasmid, orone which is replicated with the host chromosome, such as abacteriophage. Specific examples of expression vectors of the inventionare the plasmids pSPE A-R described in Example 9 below and shown in theappended FIG. 13.

In a still further aspect, the invention relates to a microorganismwhich is capable of expressing a DNA molecule or DNA fragment as definedabove and which carries a vector as described above. The microorganismis preferably a bacterium, especially a gramnegative bacterium such asE. coli.

The invention also relates to a method of producing an immunogenicdetoxified P. multocida toxin or toxin analogue, the method comprising

a) isolating a nucleotide sequence coding for the P. multocida toxin ortoxin analogue,

b) inserting said sequence, optionally in suitably modified formresulting in the expression of the detoxified toxin or toxin analogue ora subsequence coding for an immunogenic subsequence of the toxin ottoxin analogue, in an expression vector,

c) transforming a suitable host microorganism with the vector producedin step b),

d) cultivating the microorganism produced in step c) under suitableconditions for expressing the toxin or toxin analogue,

e) harvesting the toxin or toxin analogue from the culture, and

f) optionally subjecting the toxin to posttranslational modifications toproduce the detoxified toxin or toxin analogue.

In step a) of the method, the nucleotide sequence may for instance beisolated by establishing a P. multocida gene library and screening fortoxin-positive clones in accordance with established methods asindicated above as well as described in detail in Example 4 below.

In step b) of the method, the modification of the sequence optionallycarried out may be performed before or after the sequence has beeninserted in the vector. The modification may comprise substitution,addition, insertion or deletion of one or more nucleotides in thesequence or a combination thereof, as explained above.

The transformation in step c) of the method may be carried out bystandard procedures, such as disclosed in Maniatis et al. (ref. 14).

The cultivation of the host microorganism in step d) of the method maybe carried out in a culture medium conventionally used for fermentationpurposes, e.g. Luria Broth medium, and under conditions with respect topH, temperature, aeration, etc. suited to the type of microorganism inquestion, e.g. as disclosed in Maniatis et al. (ref. 14).

In step e) of the method, the harvesting of the toxin or toxin analoguemay proceed by well-known methods such as by precipitation, gelfiltration, ion exchange or HPLC reverse phase chromatography orimmunoaffinity chromatography.

If the nucleotide sequence coding for the toxin or toxin analogue hasnot been modified in step b) of the method to result in expression ofthe detoxified toxin or toxin analogue, the toxin or toxin analogueshould be subjected to posttranslational modifications in step f) of themethod, for instance thermal treatment, treatment with a chemical suchas formaldehyde, glutaraldehyde or a suitable proteolytic enzyme, e.g.trypsin, or substitution, addition, insertion or deletion of one or moreamino acids in the toxin or toxin analogue.

In a still further aspect, the invention concerns a method of producinga vaccine for immunizing an animal, including a human being, againstdiseases caused by microorganisms producing an osteolytic toxin, themethod comprising formulating the toxin or toxin analogue produced byrecombinant DNA techniques or by peptide synthesis as described abovewith an immunologically acceptable carrier or vehicle, such as thoseindicated above.

In a further, interesting aspect, the present invention relates to anon-pathogenic microorganism which carries and is capable of expressingan inserted nucleotide sequence coding for an immunogenic detoxified P.multocida toxin or toxin analogue for use as a live vaccine for theimmunization of an animal against diseases caused by microorganismsproducing an osteolytic toxin. The use of a live vaccine might beadvantageous since there is some indication that vaccines based onliving organisms show an excellent immunogenicity, often conferring alifelong immunity against the disease in question. Live vaccines alsotend to be less expensive to produce than those based on a purifiedprotein, no purification step being required.

In order to provide expression of the toxin or toxin analogue indetoxified form, the nucleotide sequence coding for the toxin or toxinanalogue may be suitably modified, either before or after introductioninto the host microorganism, by substitution, addition, insertion ordeletion of one or more nucleotides in the sequence or a combination ofthese measures, as explained above.

In a particularly advantageous embodiment of the live vaccine of theinvention, the nucleotide sequence coding for the toxin or toxinanalogue is expressed on the outer surface of the host cell. Thisprovides a favorable presentation of the toxin epitope(s) which will berecognized by the immune defense mechanisms of the animal to which thelive vaccine is administered, thus provoking an appropriate immuneresponse. One way of providing the expression of the toxin or toxinanalogue on the cell surface is to fuse the nucleotide sequence encodingthe toxin or toxin analogue to another nucleotide sequence encoding asurface protein or a subsequence thereof (e.g. a signal peptide) whichcause the toxin or toxin analogue to be expressed on the outer surfaceof the host cell, optionally as a fused polypeptide, Examples of usefulsurface proteins are adhesins, fimbrial proteins, e.g. the E. coli K88or Type 1 fimbrial protein, or the LamB protein of E. coli.

The invention also relates to the use of a recombinant, detoxifiedimmunogenic P. multocida toxin or toxin analogue for preparing a vaccinefor the immunization of an animal, including a human being, againstdiseases caused by microorganisms producing an osteolytic toxin. Thetoxin or toxin analogue used for immunization may be one encoded by theDNA sequence shown in FIGS. 10 (a)-(j) (SEQ ID NO:1) or a modificationthereof as explained above.

Similarly, the present invention relates to a method of immunizing ananimal, including a human being, against diseases caused bymicroorganisms producing an osteolytic toxin, the method comprisingadministering to the animal an immunogenically effective amount of arecombinant detoxified immunogenic P. multocida toxin or toxin analogue,such as the one encoded by the DNA sequence shown in FIGS. 10 (a)-(j)(SEQ ID NO:1) or a modification thereof. The toxin or toxin analogue maybe administered intravenously, intramuscularly, subcutaneously,intraperitoneally, orally or intranasally. It is contemplated that asuitable dosage range will be 0.1-500 μg, dependent on the age andcondition of the animal in question, the route and form ofadministration and the immunogenicity of the toxin or toxin analogue.

In a preferred embodiment, the monoclonal antibody of the presentinvention is one raised against the P. multocida toxin produced by P.multocida ssp. multocida 45/78, which is publicly available from theNational Collection of Type Cultures (NCTC), Central Public HealthLaboratory, London, England, with the accession number NCTC 12178.

In connection with the research leading to the present invention,several different monoclonal antibodies to the toxin produced by thisPasteurella strain have been prepared (vide Example 1 below),representative examples of which are the ones produced by the hybridomacell lines P3F37 and P3F51. Samples of these cell lines were depositedin accordance with the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure, on Dec. 3, 1987, in the European Collectionof Animal Cell Cultures (ECACC), Centre for Applied Microbiology andResearch, Porton Down, Salisbury, Wiltshire, Great Britain, with theaccession numbers ECACC 87120301 and ECACC 87120302, respectively.

The monoclonal antibody of the invention may be prepared by a methodcomprising:

a) Immunizing a suitable animal or animal cell with an immunogenic P.multocida toxin or toxin analogue to obtain cells producing an antibodyto said toxin or toxin analogue,

b) fusing cells producing the antibody with cells of a suitable myelomacell line, and selecting and cloning the resulting hybridoma cellsproducing said antibody, or

c) immortalizing an unfused cell line producing said antibody, e.g. byviral transformation, followed by

d) growing the cells of step b) or c) in a suitable medium to producesaid antibody and harvesting the antibody from the growth medium.

The initial immunization of the animals in step a) of the methodrequires a modification of the conventional method of producingmonoclonal antibodies disclosed by Kohler and Milstein, Nature 256,1975, p. 495, since, PMT, even when it is administered to mice insublethal doses, will cause an atrophy of the spleen which seriouslycomplicates the hybridoma technique. The animals must thereforeinitially be immunized with a detoxified toxin preparation as it hasbeen found that any subsequent booster immunizations may be carried outwith the native toxin which need not be detoxified before use. Theimmunization may otherwise be carried out according to conventionalprocedures, e.g. by administering a solution or suspension of the(detoxified) toxin or toxin analogue in a suitable solvent such asisotonic saline or phosphate buffered saline optionally containing anadjuvant such as one of those indicated above. Animals suited forimmunization purposes are, for instance, mice, rabbits, goats, sheep,guinea pigs, etc. The bleeding of the animals and recovery of thepolyclonal antibodies may be performed by conventional procedures.

The antibody-producing cells used for fusion to the myeloma cells arepreferably spleen or lymph cells (e.g. lymphoblasts, B-lymphocytes,plasma cells or related cells isolated from spleen, lymph nodes orfunctionally related tissues). The fusion of antibody-producing cellsand myeloma cells may be performed substantially as described by Kohlerand Milstein, supra, that is, preferably in the presence of a fusionpromoter such as polyethylene glycol. A ratio of about 3antibody-producing cells per myeloma cell is preferred. The myeloma cellline employed is preferably of a type unable to survive in selectivemedium and unable to produce immunoglobulins by itself; one type of cellline frequently used for cell fusions is one which lacks the enzymehypoxanthine-guanine phosphoribosyltransferase and which is consequentlyunable to grow in a medium containing hypoxanthine, aminopterin andthymidine (HAT medium).

The selection of hybridoma cells which produce an antitoxin-antibody maythen be carried out by culturing unfused antibody-producing cells,unfused myeloma cells and supposedly fused cells in a selective medium(such as HAT) in which the unfused myeloma cells cannot grow andeventually die out. The unfused antibody-producing cells can onlysurvive for a limited period of time after which they also die out. Onthe other hand, successfully fused cells continue to grow as they haveinherited permanent growth properties from the parent myeloma cells andthe ability to survive in the selective medium from the parentantibody-producing cells.

Alternatively to the production of hybridomas, immortalized unfusedantibody-producing cells may be produced either by transferring thegenes responsible for production of immunoglobulin from the hybridoma toanother (more viable) cell line or by viral transformation as describedby Klein et al., (ref. 16).

The resulting antibody-producing cells (whether hybridomas or unfusedcells) may be grown in vitro after cloning, e.g. as described in Example1 below, in a suitable medium, such as RPMI 1640. This results in theproduction of monoclonal antibodies of a very high purity as these aresecreted into the culture supernatant by the cells. The antibodies maythen be isolated by conventional methods such as centrifugation,filtration, precipitation, chromatography, or a combination thereof.

In an alternative method, the monoclonal antibodies may also be producedin the body cavity of an animal such as a mouse. In this embodiment, theantibody-producing cell is injected into an animal such as a mouseresulting in the formation of an ascites tumour which releases highconcentrations of the antibody in the ascites of the animal. Althoughthe animals will also produce normal antibodies, these will only amountto a minor percentage of the monoclonal antibodies which may be purifiedfrom ascites by standard purification procedures such as centrifugation,filtration, precipitation, chromatography or a combination thereof.

In a further aspect, the invention relates to a diagnostic agent whichcomprises a monoclonal antibody as defined above.

Although in some cases such as when the diagnostic agent is to beemployed in an agglutination assay in which solid particles to which theantibody is coupled agglutinate in the presence of a P. multocida toxinin the sample subjected to testing, no labelling of the monoclonalantibody is necessary, it is preferred for most purposes to provide theantibody with a label in order to detect bound antibody. In a doubleantibody ("sandwich") assay, at least one of the antibodies may beprovided with a label. Substances useful as labels in the presentcontext may be selected from enzymes, fluorescers, radioactive isotopesand complexing agents such as biotin.

Examples of enzymes which may be used as label substances areperoxidases, e.g. horseradish peroxidase, or phosphatases. e.g. alkalinephosphatases. As enzymes are not directly detectable, they must becombined with a substrate to form a detectable reaction product whichmay, for instance, be fluorescent or coloured. Examples of usefulsubstrates are H₂ O₂ /o-phenylene diamine, H₂ O₂/azinodiethylbenzthiazoline sulphonic acid and p-nitrophenylphosphate.

Examples of fluorescers useful as label substances are H₂ O₂/p-hydroxyphenylacetic acid and methylumbelliferyl phosphate. Suchsubstances may be detected by means of a fluorescence spectrophotometerin a manner known per se.

Examples of radioactive isotopes useful as label substances are I-125,S-35 and P-32. The radioactivity emitted by these isotopes may bemeasured in a gamma-counter or a scintillation counter in a manner knownper se.

In a favoured embodiment, the diagnostic agent comprises at least oneantibody covalently or non-covalently coupled to a solid support. Thismay be used in a double antibody assay in which case the antibodycoupled to the solid support is not labelled. The solid support may becomposed of a polymer or may comprise a matrix on which the polymer isapplied. The solid support may be selected from a plastic, e.g. latex,polystyrene, polyvinylchloride, nylon, polyvinylidene difluoride,cellulose, e.g. nitrocellulose and magnetic carrier particles (e.g. ironparticles coated with polystyrene).

For use in a diagnostic assay, the solid support may have any convenientshape. Thus, it may be in the form of a plate, e.g. a thin layer or,preferably, microtiter plate, a strip, film, paper or solid particlessuch as latex beads or the like.

Rather than being coupled directly to the solid support, the monoclonalantibody may be coupled to a ligand immobilized on a solid support.Examples of ligands include Protein A, or an immunoglobulin-specificantibody.

It should be noted that practically all methods or applications based onintact monoclonal or polyclonal antibodies could instead be performedusing fragments of the monoclonal or polyclonal antibody, e.g. F(ab')₂or Fab fragments (cf. Coding, 1978, (ref. 17).

For use in a sandwich assay, the diagnostic agent may additionallycomprise a polyclonal antibody. This antibody may be labelled and/orcoupled to a solid support as described above in connection with the.monoclonal antibody.

The monoclonal antibody of the invention may be used in a method ofdetermining the presence of a P. multocida toxin or toxin analogue in asample, the method comprising incubating the sample with a monoclonalantibody as described above and detecting the presence of bound toxin ortoxin analogue resulting from said incubation. The antibody may beprovided with a label as explained above and/or may be bound to a solidsupport as exemplified above.

In a favoured embodiment of the method, the sample is incubated with afirst monoclonal antibody coupled to a solid support and subsequentlywith a second monoclonal or polyclonal antibody provided with a label.An example of this embodiment is the sandwich ELISA (enzyme linkedimmunosorbent assay) assay described in Example 2 below.

In an alternative embodiment (a so-called competitive binding assay),the sample may be incubated with a monoclonal antibody coupled to asolid support and simultaneously or subsequently with a labelled P.multocida toxin or toxin analogue the latter competing for binding siteson the antibody with any toxin or toxin analogue present in the sample.

The sample subjected to the present method may be any sample suspectedof containing a P. multocida toxin or toxin analogue. Thus, the samplemay be selected from bacterial suspensions, bacterial extracts, culturesupernatants, animal body fluids (e.g. serum, colostrum or nasal mucous)and intermediate or final vaccine products.

Apart from the diagnostic use of the monoclonal antibody of theinvention, it is contemplated to utilize the well-known ability ofcertain monoclonal antibodies to inhibit or block the activity ofbiologically active antigens by incorporating the monoclonal antibody ina composition for the passive immunization of an animal, including ahuman being, against diseases caused by microorganisms producing anosteolytic toxin, which comprises a monoclonal antibody as describedabove and a suitable carrier or vehicle. The composition may be preparedby combining an effective immunizing amount of the antibody or fragmentthereof with a suitable carrier or vehicle. Examples of suitablecarriers and vehicles may be the ones discussed above in connection withthe vaccine of the invention. The composition may further comprise anadjuvant such as one of those indicated above.

Based on experiments with mice (cf. Example 11 below) where themonoclonal antibody induced passive immunity against PMT, it iscontemplated that a PMT-specific antibody may be used for prophylacticor therapeutic treatment of atrophic rhinitis in pigs. It may beadministered intravenously, subcutaneously or intramuscularly as well asorally in suitably protected form or by means of an intranasal aerosol.

A further use of the monoclonal antibody of the invention is in a methodof isolating a P. multocida toxin or toxin analogue, the methodcomprising adsorbing a biological material containing said toxin ortoxin analogue to a matrix comprising an immobilized monoclonal antibodyas described above, eluting said toxin or toxin analogue from saidmatrix and recovering said toxin or toxin analogue from the eluate.

The matrix may be composed of any suitable material usually employed foraffinity chromatographic purposes such as agarose, dextran, controlledpore glass, DEAE cellulose, optionally activated by means of CNBr,divinylsulphone, etc. in a manner known per se.

The present invention further relates to a diagnostic agent for thedetection of PMT-producing microorganisms, which comprises a labelledDNA sequence homologous with a DNA sequence coding for a Pasterurellamultocida toxin or toxin analogue. In this context, the term "homologouswith" is intended to indicate that the DNA sequence comprises at leastone stretch of deoxyribonucleotides of at least 15 bases with 80%homology to a part of the shown sequence or to a part of the sequenceencoding a toxin analogue.

In a method employing the diagnostic agent, probe DNA is labelled, andthe DNA is denatured to separate the strands in both probe and sampleDNA; the DNAs are mixed and the strands are left to reform the doublehelical structure, but in case of homology, some of the probe DNA willhave combined with the sample DNA. This is known as hybridization and isdescribed by for instance Southern, 1980, (ref. 18). The DNA used as theprobe should have a unique nucleotide sequence of a certain length inorder to be sufficiently specific as a diagnostic agent. The probe DNAmay advantageously be labelled with a radioactive isotope such as H-3,I-125, S-35 or P-32 as described e.g. by Rigby et al., 1977, (ref. 19);a complexing agent such as biotin (Gebeyechu et al., 1987, (ref. 20); orwith digoxygenin-dUTP according to the method described by themanufacturer of the reagent, Boehringer, Mannheim.

In a particular embodiment of the invention, detection of the presenceof Pasteurella multocida toxin producing microorganisms in a sample isperformed by use of a DNA probe in the polymerase chain reactionprocedure described by Saiki et al., 1985, (ref. 21). The polymerasechain reaction (PCR) is a procedure used for the amplification of DNApresent in a sample. The procedure involves the use of twooligonucleotide primers which flank the DNA segment to be amplified. Theoligonucleotide primers may e.g. comprise the regions of the gene codingfor Pasteurella multocida toxin or toxin analogue and may thus be usedto amplify the said gene or part of it present in a sample. Theoligonucleotide primers hybridize to opposite strands of the DNAsequence to be amplified, and the primers are extended by using DNApolymerase, e.g. the Klenow fragment of E.coli DNA polymerase I oranother useful DNA polymerase such as the Taq DNA polymerase, so as tosynthesize a DNA sequence which is complementary to the DNA sequence towhich the primers are annealed. Subsequent to the synthesis of thesecomplementary sequences, the DNA synthesized is denatured, e.g. byheating, from the "parent DNA strings", and the parent strings as wellas the newly synthesized DNA strings are subjected to a new PCRamplification cycle. In this manner, it is possible to obtain asubstantial amplification of specific DNA sequences which are present ina sample. By use of the PCR amplification method, it may be possible toamplify and then detect originally very small and undetectable amountsof DNA sequences present in a sample.

In a still further aspect, the present invention relates to a method ofdetermining the presence of antibodies against a P. multocida toxin ortoxin analogue in a sample, the method comprising incubating the samplewith a P. multocida toxin or toxin analogue and detecting the presenceof bound antibody resulting from said incubation.

A diagnostic agent comprising the toxin or toxin analogue used in thismethod may otherwise exhibit any of the features described above fordiagnostic agents comprising the monoclonal antibody and be used insimilar detection methods although these will detect bound antibodyrather than bound toxin as such. The diagnostic agent may be useful, forinstance as a reference standard or to detect anti-toxin antibodies inbody fluids, e.g. serum, colostrum or nasal mucous, from animals exposedto the toxin or toxin analogue.

A still further use of a P.multocida toxin or toxin analogue is for thepreparation of a toxin reference standard which may be useful as astandard of comparison in qualitative or quantitative analyticalprocedures. In a qualitative procedure, the standard toxin in a knownconcentration may be reacted with a monoclonal or a polyclonal antibodyraised against the toxin or toxin analogue, a positive reactionindicating the specificity of the antibodies. In another aspect, thereference standard preparation is applied in a quantitative analyticalprocedure by which different concentrations of the preparation isreacted with a monoclonal or a polyclonal antibody in order to provide acalibration curve which may allow the precise amount of toxin or toxinanalogue in a sample to be estimated.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further disclosed in the following with reference tothe drawings in which

FIG. 1 is a graph showing the titration of PMT in a quantitativesandwich ELISA. The absorbance at 492 nm obtained in the ELISA isplotted against the PMT concentration. The minimum detectableconcentration of PMT is about 1 ng/ml corresponding to about 50 μg or0.35 fmol.

FIG. 2 shows an SDS-PAGE of fractions from the affinity chromatographydescribed in Example 3. Lane A: the culture supernatant applied on thecolumn, lane O: the effluent from the column, lane E: the elutedpurified PMT, and lane M: molecular weight marker proteins,

FIG. 3 is a Western blot showing PMT production by the 5 positiverecombinant E. coli clones detected in the screening procedure. Lane 1:SPE 301; lanes 2 and 3: SPE 308; lane 4: SPE 315; lanes 5 and 6: SPE312; lane 7: SPE 311; lane 8: purified PMT.

FIG. 4 is a restriction enzyme cleavage map of the plasmid pSPE 308 witha length of 21.5 kb (kilobase pairs). The hatched area denotes P.multocida DNA, the shaded area denotes the pmt gene and the verticallyhatched area denotes plasmid pUN121 DNA.

FIG. 5 is a restriction enzyme cleavage map of the plasmid pSPE 312 witha length of 13.8 kb. The hatched area denotes P. multocida DNA, theshaded area denotes the pmt gene and the vertically hatched area denotesplasmid pUN121 DNA.

FIG. 6 is a restriction enzyme cleavage map of plasmids constructed byenzymatic cleavage of the plasmid pSPE 308. The hatched area denotes P.multocida DNA, the shaded area denotes the pmt gene, and the verticallyhatched area denotes pUN121 DNA.

FIG. 7 is a restriction enzyme cleavage map of plasmids constructed byenzymatic cleavage of pSPE 312. The hatched area denotes P. multocidaDNA, the vertically hatched area denotes pUN121 DNA, and the shaded areadenotes the pmt gene.

FIG. 8 is a Western blot showing PMT production by derivatives ofplasmids pSPE 308 and pSPE 312. Lane 1: purified PMT; lane 2: pSPE 350;lane 3: pSPE 349; lane 4: pSPE 341; lane 5: pSPE 345; lane 6: pSPE 312;lanes 7 and 8: purified PMT. Plasmid pSPE 349 is identical to plasmidpSPE 347 shown in FIG. 7.

FIG. 9 is a restriction enzyme cleavage map of the pmt gene. The shadedarea denotes the pmt gene, the vertically hatched area denotes aprobable promoter, and the hatched area denotes a probable terminator.

FIGS. 10 (a)-(j) shows the DNA sequence (SEQ ID NO:1) of the pmt generegion and the amino acid sequence (SEQ ID NO:2) deduced on the basis ofthe DNA sequence. The amino acids are identified with single-lettercodes according to conventional usage. The amino acid sequence has beenshown to start at position 213 or 219.

FIG. 11 is a restriction enzyme cleavage map of the plasmid pSPE 525with a length of 7.7 kb. The hatched area denotes P. multocida DNA, theshaded area denotes the pmt gene, and the vertically hatched areadenotes pUN121 DNA.

FIG. 12 is a restriction enzyme cleavage map of the expression vectorpSPE 481 with a length of 8.25 kb. The hatched (towards the right) areadenotes P. multocida DNA, the shaded area denotes the pmt gene, thehatched (towards the left) area denotes λP_(L) DNA, the cross-hatchedarea denotes the amp gene, and the vertically hatched area denotes theorigin of replication.

FIG. 13 is a restriction enzyme cleavage map of the toxA coding region.Extension of coding region present on each derivative plasmid (pSPE A-R)is indicated (A-R) by bars. Hatched bars: Coding region in correctreading frame; open bars: Coding region not in frame with the 5' part ofthe coding region.

FIG. 14 is a Western blot showing the recognition by a mouse anti-PMTantiserum of PMT derivatives produced by the plasmids pSPE A-L. Lanes 7,13, 14 and 15: different strains harbouring the entire pmt gene; lane 1:derivative A; lane 2: derivative I; lane 3: derivative B; lane 4:derivative J; lane 5: derivative L; lanes 6 and 9: derivative E; lane 8:derivative C; lane 10: derivative G; lane 11: derivative H; lane 12:derivative D. Approximate sizes (in kilodalton) of prominent full-lengthderivatives and degradation products are indicated.

FIG. 15 is a graph showing the distribution of relative absorbances(A/A_(o)) by PMT-ELISA of extracts of non-cytopathic (hatched bars) andcytopathic (black bars) field isolates of P. multocida diluted 1:1 inPBS-T-BSA.

FIG. 16 is a graph showing the mean±SD of relative absorbances (A/A_(o))of dilutions of extracts of cytopathic (black squares) andnon-cytopathic (open squares) field isolates of P. multocida.

FIG. 17 is a graph showing the presence of anti-PMT-antibodies in serumsamples from anti-PMT-antibody-negative, infected and vaccinated pigsdetected by competive ELISA. The graph shows the 50% blocking titers atan absorbance of 492 nm.

. . negative

- - - infected

₋₋₋₋ vaccinated

FIG. 18 shows the colony hybridization of P. multocida isolates, testing17 toxin-positive and 18-toxin-negative strains as determined by theELISA and EBL cell tests for the presence of the pmt gene.

FIG. 19 shows the determination of toxic activities of cell-freesonicates of recombinant E.coli clones. E.coli strain MT102 with PUN121had no cytopathic effect on EBL-cells when diluted 1/25 in PBS (a).Sonicates of E.coli SPE312 (b) and toxigenic P.multocida (NCTC 12178)(c) diluted 1/3125 showed significant and identical effects(80×magnification).

FIG. 20 shows the P.multocida DNA flanking the pmt-gene (black area).The extension of the inserts of the plasmids pSPE308, pSPE312, pSPE344,pLOA03 and pLOB03 are indicated. The DNA contained in the probes usedfor the blotting (slant hatched area), and the fragments which containthe two homologous sequences (vertically hatched area) are shown.

FIG. 21 shows a Southern blot of restriction enzyme digested P.multocidaDNA. Probe: 2,4 kb BglII-EcoRI fragment of pLOB03. Lanes 10*-14* is ashort time exposure of lanes 10-14.

Lanes 1-4: Toxigenic P.multocida 45/78. Lanes 5-9: NontoxigenicP.multocida MH81P8. Lane 10: pSPE308. Lane 11: pLOA03. Lane 12: pLOA02.Lane 13: pSPE312. Lane 14: pLOB03.

Restriction enzymes used: HindIII: Lanes 1, 5, 10, 11, 12 and 13.

EcoRI: Lanes 2, 6 and 14. BglII: Lanes 3 and 7. PvuII: Lanes 4 and 8.

PstI: Lane 9.

FIG. 22 shows a dot blot of 24 different P.multocida bacteriophagegenomes. Probe: pLOA03. The probe does not hybridize to B2 and C5. A7:pSPE308; B7: pSPE312, C7: pLOA03 and D7: pLOB03 are positive controls.

EXAMPLE 1

Preparation of monoclonal antibodies against P. multocida toxin

Immunization

P. multocida toxin (PMT) was purified as described by Foged et al. (ref.6), i.e., by 50% (NH₄)₂ SO₄ -precipitation of a cell-free extract of thetoxigenic type D strain of P. multocida ssp. multocida 45/78 (refs. 3,22) followed by DEAE-Sephacel® chromatography and preparativepolyacrylamide gel electrophoresis (PAGE) in a manner known per se.

A suspension of the (NH₄)₂ SO₄ -precipitate prepared as described aboveand containing approx. 25 μg/ml of PMT was detoxified by incubation with0.37% formaldehyde at 37° C. for 1 h. Female BALB/c mice (8-12 weeksold) were immunized subcutaneously on day 0 and 14 with 300 μl of a 1:1dilution of the crude preparation of detoxified P. multocida toxin andFreund's incomplete adjuvant (day 0) or PBS (day 14). On day 30 and 45 1μg of native PMT in 200 μl PBS was injected subcutaneously and on day 60the mice were boosted intravenously with 0.5 μg of PMT in 100 μl PBS.Three days after the booster injection the mice were sacrified and theirspleens removed for fusion.

Production of hybridoma cell lines and monoclonal antibodies

According to procedures described by Fazetas et al. (ref. 23) andGebeychu et al. (ref. 20), the spleen cells and P3-X63-Ag8.653 myelomacells were fused using 50% PEG 4000 GK (Merck) and the resultinghybridoma cells were grown selectively inhypoxanthine/aminopterin/thymine (HAT)--supplemented RPMI 1640 mediumcontaining 15% fetal calf serum (FCS) and 4% human endothelial cellsupernatant (Costar, The Netherlands).

Hybridoma cell lines were selected by analyzing their respectivemonoclonal antibodies by ELISA and immunoblotting.

ELISA for detection and titration of monoclonal antibodies

Microtiter plates (96-well Immuno Plate II, Nunc, Denmark) were coatedwith 50 μl/well of a 0.75 μg/ml solution of purified PMT in PBS at 4° C.for 16 h. and at 20° C. for 1 h. The wells were emptied and blocked with200 μl PBS-T-BSA (PBS containing 0.05% (v/v) Tween®20 and 1% (w/v)bovine serum albumin) per well at 20° C. for 1 h., then washed 3 timeswith PBS-T. Fifty μl/well of hybridoma culture supernatant was appliedat 20° C. for 1 h., and the plates were washed as described above. Theanti-PMT antibody activity was measured calorimetrically afterincubating at 20° C. for 1 h. with 50 μl/well of sheep anti-mouseimmunoglobulin conjugated with horseradish peroxidase (AmershamInternational, U.K.) diluted 1:1,500 in PBS-T-BSA and (after 3 furtherPBS-T washes as above) with 50 μl of an o-phenylene diamine (OPD)H₂ O₂substrate solution. The reaction was stopped with 150 μl of 2 M H₂ SO₄after 5 min. and absorbance was determined in a Kontron SLT-210photometer (SLT Lab-instr., Zurich, Switzerland) at 492 nm (ref. 620nm).

The mean absorbance at the saturation level of the titration curve(A_(sat)) and the mean concentration of the MAb that resulted in 50% ofthe A_(sat) (C₅₀ %) was determined by ELISA as described above, exceptthat serial dilutions of the protein-A purified MAb in PBS-T-BSA wasused. All results were based on at least duplicate determinations.

Immunoblotting

To determine the specificity of the monoclonal antibodies, the proteinscontained in a crude cell-free extract of P. multocida 45/78, wereseparated by SDS-PAGE before transfer to a nitrocellulose membrane andimmunological detection. Polyacrylamide gels (total acrylamide: 10%,relative bis-acrylamide: 3%) and an electrophoresis buffer were preparedaccording to Laemmli (ref. 24). Electrophoresis was performed verticallyat 10° C. at a constant voltage of 60 V for 16 h. or 250 V for 4 h.Protein-bands on gels were either visualized by silver staining with adetection limit of less than 1 ng of protein per band (8) or transferredto a nitrocellulose membrane (0.45 μm) using a semidry electroblotter(Ancos, .O slashed.lstykke, Denmark (9)). The proteins on thenitrocellulose membrane were either detected by a colloidal gold silverenhancement staining method (detection limit: approx. 0.5 ng of proteinper band) (ref. 25) or immunologically by a modification of the methodpreviously described by Bjerrum et al. (ref. 26). A positive reaction inimmunoblotting was recorded as + or (+), when an intense (or weak)staining of the PMT-band but no other protein band was observed.Staining of other bands or no reaction was recorded as -.

The molecular weight of PMT was estimated by comparison with knownmarkers: ovalbumin (43.0 kd), BSA (66.3 kd), phosphorylase B (97.4 kd),β-galactosidase (116.2 kd), RNA-polymerase β (150.6 kd) and β' (155.2kd) and myosin (approx. 200 kd).

ELISA for estimating epitope specificity

Estimation of apparent epitope specificity of anti-PMT MAbs was done bya competitive ELISA similar to a method described by Anderson et al.(ref. 27). Microtiter plates were coated with PMT and blocked asdescribed above. Fifty μl of the competing MAb diluted to 10 μg/ml inPBS-T-BSA was added and incubated for 1 h. at 20° C. Without aspirationof the wells 25 μl biotinylated monoclonal antibody was added and themixture was incubated for 20 min. at 20° C. After washings 50 μl of a1:2,500 dilution of horseradish peroxidase-conjugated avidin(Kem-En-Tec, Denmark) was added and the plates incubated for 45 min. at20° C. The substrate, reaction time and determination of absorbance wereas described above.

The biotinylated MAb was used at a working dilution resulting in approx.75% of the absorbance at the saturation level on the titration curve.This curve was obtained by using a diluent instead of the competing MAband serial dilutions of the biotinylated MAb. The extent of blocking bya competitive MAb was calculated according to the formula(1-A/A_(o))×100%, where A is the mean of the absorbance for three wellswith the competing MAb and A_(o) is the mean of the absorbance of eightwells containing diluent instead of the competing MAb.

The data of 10 representative monoclonal antibodies (MAbs), all of theIgG₁ subclass, out of 92 ELISA-positive supernatants are shown in Tables1 and 2.

                  TABLE 1                                                         ______________________________________                                        Characterization of 10 representative MAbs                                    Hybridoma                                                                              Represen-          C.sub.50%                                         group No.                                                                              tative MAb                                                                              A.sub.sat                                                                              ng/ml                                                                              Immunoblotting                               ______________________________________                                        1        P3F51     1.2      110  +                                            2        P3F64     0.4      250  +                                            3        P3F37     0.7      30   (+)                                          4        P4F58     0.7      110  +                                            5        P3F22     0.6      35   +                                            6        P4F46     1.3      55   +                                            7        P4F38     1.9      40   +                                            8        P4F55     1.3      33   +                                            9        P3F50     1.8      315  +                                            10       P3F53     0.9      300  (+)                                          ______________________________________                                         .sup.a) A.sub.sat is the mean absorbance at 492 nm at the saturation leve     in the ELISA titration.                                                  

                                      TABLE 2                                     __________________________________________________________________________    Extent of blocking by 10 representative MAbs                                  in the competitive ELISA                                                      Competing MAb                                                                 (hybridoma Biotinylated detector MAb (% decrease in A.sub.o.sup.a)            group No.  1  2  3  4  5  6  7  8  9  10                                      __________________________________________________________________________    P3F51   (1)                                                                              92 b                                                               P3F64   (2)   95 44                                                           P3F37   (3)   63 95 78                                                        P4F58   (4)      91 96 73                                                     P3F22   (5)         71 88                                                     P4F46.sup.c                                                                           (6)               92 90 93 16                                         P4F38.sup.c                                                                           (7)               93 92 95 27                                         P4F55.sup.c                                                                           (8)               92 92 95 15                                         P3F50   (9)               16 24 13 84 91                                      P3F53   (10)                       56 83                                              A.sub.o                                                                          1.43                                                                             0.19                                                                             0.53                                                                             0.80                                                                             0.64                                                                             0.64                                                                             0.85                                                                             1.01                                                                             0.26                                                                             0.52                                    __________________________________________________________________________     .sup.a A.sub.o  is the mean absorbance with diluent instead of competing      MAb                                                                           .sup.b No blocking (between 12% increase and 9% decrease in A.sub.o)          .sup.c The closely related hybridoma groups 6, 7 and 8 were differentiate     by a twosite competitive ELISA using a catching MAb (method not               described). Results indicated that group 6 was related to groups 3 and 4,     group 7 to no other groups and group 8 to group 1.                       

The selected hybridoma cell lines were then cloned until they werestable. The resulting clones were then grown in "cell factories" (Nunc,Denmark) at 37° C. in RPMI 1640 medium supplemented with 10% FCS as wellas injected about 5×10⁶ cells/mouse into Balb/c mice which after acertain incubation time leads to the formation of a tumour in theperitoneum of the mouse releasing high quantities of antibody in itsascites (about 5-10 ml containing 5-25 mg/ml).

The hybridoma cell culture supernatants were passed through a protein Aagarose column (Kem-En-Tec, Denmark). Bound antibodies were eluted with0.05M acetic acid, pH 4.0, or 0.03M citric acid, pH 3.0, and immediatelyneutralized with an appropriate buffer. Purified antibodies werebiotinylated as described by Guesdon et al., 1979, (ref. 28).

Two hybridoma cell lines, P3F37 and P3F51 shown in Table 1 to produceMAb, were deposited on Dec. 3, 1987 in the European Collection of AnimalCell Cultures, Centre for Applied Microbiology and Research, PortonDown, Salisbury, Wiltshire SP4 OJG, U.K., with the Accession numbersECACC 87120301 and ECACC 87120302, respectively.

EXAMPLE 2

Quantification of PMT

Quantification of PMT was carried out by a sandwich ELISA procedure. Thesandwich ELISA was initiated by coating each well of a microtiter plate(96 wells Immuno Plate II, Nunc, Denmark) with 50 μl of 2 μg/ml of theanti-PMT MAb, P3F51 (produced in Example 1) in 0.05 M carbonate buffer,pH 9.6 for 16 hours at 4° C. and 1 hour at 20° C. Each well wasincubated for 1 hour with 200 μl of phosphate-buffered saline containing0.05% Tween 20 and 1% bovine serum albumine (PBS-T-BSA). The platescould be stored for at least 6 months by applying 20 μl/well ofPBS-sorbitol and sealing with adhesive tape. The analysis was initiatedby two PBS-T washings followed by incubation of 50 μl/well of solutionsexpected to contain PMT. The solutions were appropriately diluted inPBS-T-BSA and incubated for 1 hour at 20° C. After 3 PBS-T washings eachwell was incubated with 50 μl of 0.5 μg/ml of the biotin conjugated MAb,P3F37, for 1 hour at 20° C. followed by another 3 PBS-T washings andincubation with 50 μl/well of a 1:2,500 dilution ofhorseradish-peroxidase conjugated avidin (Kem-En-Tec, Denmark) for 45min. at 20° C. Finally, 50 μl/well of an o-phenylene diamine/H₂ O₂solution was added. The reaction was stopped with 2M H₂ SO₄ after 5 min.and absorbance was determined in an Kontron SLT-210 photometer (SLTLabinstr., Zurich, Switzerland) at 492 nm (ref. 620 nm).

Calibration was performed with a PMT-preparation quantified by aminoacid analysis (ref. 6) and all quantitative data were means of at leastdual determinations.

Using a sandwich ELISA, with the MAb P3F51 as catching antibody andbiotinylated MAb P3F37 as detecting antibody, it was possible to detectless than 50 μg of PMT in a 50 μl sample. PMT at a concentration of 1ng/ml resulted in an A₄₉₂ of approx. 0.1 corresponding to more than 8times the background absorbance (cf. FIG. 1).

EXAMPLE 3

Affinity purification of PMT

About 100 mg of the protein-A purified MAb P3F51 prepared as describedin Example 1 was coupled to 40 ml divinyl sulphone agarose (Mini-Leak,Kem-En-Tec, Denmark) as described by the manufacturer and loaded on acolumn (2.5×10 cm). The supernatant obtained by cultivation of thetoxigenic type D strain P. multocida 45/78 was centrifuged (12,000×g for30 min. at 4° C.), filtered (Gelman, 0.45 μm), mixed with 1/10 vol. of 1M Tris-HCl, pH 7.7 and NaCl was added to 0.5 M before application to theaffinity column. Repeated washings before elution of the column werecarried out with an 0.1 M Tris-HCl buffer containing first 1% Triton®X-100, then 1.5 M NaCl and finally 0.1 M NaCl. All washing bufferscontained 0.1% sodium azide and had a pH of 7.8. The PMT was eluted by0.1 M glycine-HCl, pH 2.8 and immediately neutralized with 1 M K₂ HPO₄,pH 9.0.

The presence of PMT in the culture supernatant applied to the affinitycolumn was indicated by the approx. 143 kd protein band seen by SDS-PAGE(FIG. 2). The staining pattern of proteins in the material passingthrough the column (i.e. the effluent) was identical to that seen withmaterial before application, except for the 143 kd protein, which wasretained on the column. Accordingly, the approx. 143 kd protein band isthe only staining seen when the protein composition of the elutedmaterial is visualized by SDS-PAGE (FIG. 2).

Approx. 2.67 mg of the 3.41 mg of PMT applied to the column was elutedin a final volume of 8 ml resulting in a 78% yield by affinitychromatography (Table 3). Nearly all the remaining 22% of PMT appliedwas eluted in fractions with PMT-concentrations below 50 μg/ml.

The specific purity (μg PMT/mg protein) was 284 times higher in theeluted material than in the culture supernatant (Table 3).

The average minimal dermonecrotic dose of affinity purified PMT inguinea pigs after intradermal injection and the average MCD of PMT forembryonic bovine lung (EBL) cells in the standardized EBL-cell test(ref. 29) was approx. 35 ng and 30 μg, respectively. The LD₅₀ of PMT inBALB/c mice was 20 to 40 ng (corresponding to approx. 1.5 μg/kg) and inWistar rats approx. 100 ng (corresponding to 0.5 μg/kg) whenadministered intraperitoneally in a single dose.

                  TABLE 3                                                         ______________________________________                                        Purification of PMT by affinity chromatography                                        Vol.  Protein PMT     Purifica-                                                                            Recovery of                                      (ml)  (mg)    (mg)    factor PMT, %                                   ______________________________________                                        Applied culture                                                                         13,200  970     3.41  1      100                                    supernatant from                                                              P. multocida                                                                  45/78                                                                         Effluent from                                                                           13,200  970     <0.01 N.D..sup.a                                                                           <0.35                                  affinity column                                                               Eluted material                                                                            8    N.D.    2.67  284.sup.b                                                                            78                                     ______________________________________                                         .sup.a) N.D. not determined                                                   .sup.b) Estimated on the assumption of purity of PMT in the eluted            material.                                                                

EXAMPLE 4

Establishment of a P. multocida gene library in Escherichia coli

Donor strain

P. multocida strain 45/78. The strain produces a dermonecrotic toxin asdescribed by Foged et al. (ref. 6).

Recipient strain

Escherichia coli K-12 strain MT102. Genotype: thi, araD139,(ara-leu)Δ7697, lacΔX74, galU, rpsL, hsdR. This strain was constructed(by Mogens Trier Hansen, Novo Industri A/S, Denmark) as follows:##STR1## Escherichia coli strain list (all strains are K-12 strains)

    ______________________________________                                                                      REFERENCE                                       STRAIN GENOTYPE               OR SOURCE                                       ______________________________________                                        MC1000 araD139, Δ(ara-Leu)7697, ΔlacX74, galU,                                                  a)                                                     galK, rpsL.                                                            S.o slashed.1386                                                                     leu, Δ(deoO-deoC), Tn10-6 (91% co-trans-                                                       b)                                                     ducible with deo                                                       MT85   araD139, Δ(ara-leu)7697, ΔlacX74, galU,                                                  c)                                                     galK, rpsL, T10-6, Δ(deoO-deoC)                                  ≅804                                                                       met, supF, r-, m+, Δ(gal-lac)                                                                  d)                                              ______________________________________                                         b) from Bente Mygind, Laboratoriet for Biologisk Kemi B, University of        Copenhagen, Denmark.                                                          c) constructed by Mogens Trier Hansen as shown above.                         a) Casadaban and Cohen, 1980, (ref. 30).                                      d) Wood, 1966 (ref. 31).                                                 

Media

P. multocida was grown in Tryptic Soy Broth purchased from DIFCO. E.coli was grown in LB medium (ref. 27).

Restriction enzymes and T4 DNA ligase were obtained from New EnglandBiolabs and used as recommended by the manufacturer.

DNA extraction

To isolate chromosomal DNA from P. multocida strain 45/78, cells from a250 ml overnight stationary culture were resuspended in 10 ml 50 mMTris-HCl, pH 8.0, 100 mM EDTA, and incubated with 25 mg lysozyme for 20minutes at 37° C. 2 ml of 10% (w/v) SDS were added to the mixture whichwas mixed and put on ice for 10 minutes. To the solution was then added15 ml of phenol saturated with TE-buffer (10 mM Tris-HCl, pH 8.0, 1 mMEDTA), after which it was heated to 65° C., mixed gently and cooled onice. After centrifugation for 30 minutes at 4000×g the aqueous phase wasextracted with ether and ethanol precipitated, and the pelletresuspended in TE-buffer. The DNA was further purified by banding in aCsCl density gradient (ref. 27). After purification, the DNA wasresuspended in 1 ml TE-buffer.

Preparation of clonable DNA fragments

9-22 kilobase pair (kb) DNA fragments with 5'GATC overhangs wereprepared in the following way. Chromosomal DNA prepared as describedabove was digested partially by incubation with the restrictionendonuclease Sau3A. At certain intervals after the incubation had beeninitiated, fractions of the incubation mixture were stopped with 1/20volume 0.25 M EDTA. A sample of each fraction was run in a 1% agarosegel in TAE buffer as described in (ref. 27), and a fraction containing4-22 kb fragments was identified. This fraction was further fractionatedon a 8 ml sucrose gradient (40-10%) by layering the DNA on top of thegradient prior to ultracentrifugation at 41000 rpm for 7.5 hours. 0.5 mlsubfractions were extracted, diluted with 1 volume TE-buffer,ethanol-precipitated and resuspended in TE-buffer. Two of these,containing 9-16 and 15-22 kb fragments respectively, were used in thefollowing cloning steps.

Cloning procedure

9-16 kb and 15-22 kb DNA fragments with 5'GATC overhangs were ligatedwith BclI restricted pUN121 (refs. 27 and 19) by means of T4 DNA ligase.Insertion of DNA into the unique BclI site of this vector leads toinactivation of the cI gene, encoding the lambda cI repressor, whichsubsequently is unable to repress transcription from the plasmid-encodedλ PL promoter into the tetracycline resistance gene. The resultingplasmids were tranformed to competent E. coli MT 102 cells as describedin (ref. 27). Positive selection for clones with plasmid inserts isachieved by adding tetracycline to the medium (10 μg/ml). Using standardtransformation techniques (ref. 27), 3332 tetracycline resistantrecombinant E. coli clones were obtained,--100% of them containinginserts, thus constituting a P. multocida strain 45/78 gene library inE. coli. Colonies of the E. coli clones were grown on LB-platescontaining 10 μg/ml of tetracycline. A scrape of these colonies wasstored at -80° C. in a 20% glycerol solution.

EXAMPLE 5

Identification of P. multocida toxin producing E. coli clones

Screening procedure

The gene library was screened using the colony blot method to transfercolonies to nitrocellulose (ref. 14).

PMT-producing clones were then detected by incubation of thenitrocellulose filters as follows: A) 15 min. in 50 mM Tris, pH 9.6, 150mM NaCl, 0.05% Tween-20 (washing buffer) and 2 μg/ml DNaseI, B) 2×10min. in washing buffer without DNaseI, C) 30 min. in washing buffer and3% gelatine and heated, D) 2×10 min. in washing buffer with 1% TritonX-100, E) 60 min. in a 10-fold dilution in washing buffer of apreviously described hybridoma supernatant P3F51, F) 3×5 min. in washingbuffer, G) 60 min. in washing buffer with horseradish peroxidaselinked-anti-mouse Ig whole antibodies from Amersham (NA.931) diluted1:1000, H) 3×5 min. in washing buffer,₁) 1 min. in 10 mM Na₂ HPO₄, 10 mMcitric acid, pH 5.0 (C/P buffer), and J) approximately 5 min. in astaining solution mixed immediately prior to use consisting of 80 mgdioctylsodiumsulfosuccinate (DONS), 24 mg3,3',5,5'-tetramethylbenzidine, 10 ml ethanol, 30 ml C/P buffer and 20μl H₂ O₂. The enzyme reaction was terminated by incubation in 100 mgDONS in 12.5 ml ethanol and 37.5 ml H₂ O.

The following clones were found to be positive by this procedure: SPE301, 308, 311, 312 and 315.

Western blot

The positive clones obtained in the screening procedure were furtheranalyzed using the Western blot technique (ref. 32).

In the Western blot procedure, one ml overnight cultures were pelleted(6000×g for 5 min.) and resuspended in 0.5 M Tris-HCL, pH 6.8, 3% SDS,15% glycerol, 5% mercaptoethanol and bromophenol blue. The samples wereboiled for 5 minutes prior to loading on a gel. Proteins were separatedby sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)(11), the separating gel consisting of 7% (w/v) acrylamide(acrylamide/bisacrylamide ratio of 40:1) in 0.4 M Tris, pH 6.8, 0.1%SDS, 0.05% glycerol.

Subsequent transfer to nitrocellulose filters was performed in asemi-dry electroblotter as described by Kyhse-Andersen (25). Furtherhandling of the filters was as described above in the screeningprocedure.

The results appear from FIG. 3 which shows a Western blot of the 5positive clones.

The transformant strains SPE 308 (lanes 2 and 3) and SPE 312 (lanes 5and 6) harbouring the plasmids pSPE 308 and pSPE 312, respectively, wereshown to be PMT-producing, whereas SPE 301, SPE 315 and SPE 311 wereshown not to be. Purified PMT (lane 8) was used as control. Therecombinant toxin produced by SPE 308 and SPE 312 shows the sameproperties as the native protein with respect to recognition in thecompetitive ELISA as described in Example 1; the size and the toxicactivity in the EBL test (ref. 29) are unchanged.

EXAMPLE 6

Restriction mapping and localization of the PMT gene

The plasmids pSPE 308 and pSPE 312 were analyzed for recognitionsequences for restriction enzymes (restriction sites), testing forrestriction sites for most of the known enzymes with a 6 base pair (bp)recognition sequence. The results are shown in FIG. 4 (pPSPE 308) andFIG. 5 (pSPE 312). It appears from the figures that the two plasmidscarry an approximately 6 kb overlap (from position 11000 to 17000 inpSPE308). The pmt gene is therefore localized within this area.

By constructing end-point deletions in the overlapping areas in pSPE 308and pSPE 312 a number of plasmids, the pSPE 308 derivatives pSPE 336,pSPE 341, pSPE 344 and pSPE 350 as well as the pSPE 312 derivatives pSPE338, pSPE 343, pSPE 345, pSPE 347/349 and pSPE 525 were constructed. Theextents of the resulting plasmids are shown in FIGS. 6 and 7.

These plasmids were transformed to E. coli strain MT 102 and analyzedfor the production of PMT by Western blotting as described above.

In the Western blot, a single protein with an apparent molecular weightof 125000 daltons was found. The blot is shown in FIG. 8. The plasmidpSPE 349 coding for this protein is deleted of a region to the right ofthe EcoR1 site at position 8200 in pSPE 312 (cf. FIGS. 5 and 7). Thegene product of plasmid pSPE 349 therefore localizes the position andorientation of the pmt gene (shown as shaded area in FIG. 5). The codingregion begins about 3.3 kb upstream of the EcoR1 site at position 8200in PSPE 312 i.e. around the ClaI site at position 4900. Since the totalcoding region is estimated to be about 3.9 kb the structural gene endsabout position 8800 on the map shown in FIG. 5.

EXAMPLE 7

Sequencing of the pmt gene

The nucleotide sequence carrying the pmt gene as localized in Example 4was determined using the method described by Sanger et al. (ref. 33).The sequence of 4380 consecutive bp were determined. The DNA sequence ofthe region is shown in FIGS. 10 (a)-(j) (SEQ ID NO:1) in which thededuced amino acid sequence (SEQ ID NO:2) is indicated above the DNAsequence starting at position 219. The methionine codon of position219-221 is preferred as a starting codon to the methionine codon ofposition 213-218 due to its perfect spacing to the putative ribosomebinding site at position 201-210. The region containing the pmt gene wassubjected to more detailed restriction mapping by a computer search forall restriction sites for restriction enzymes with 6 bp recognitionsequences.

The results are shown in FIG. 9 which shows a high degree of conformitywith the previously constructed restriction map.

EXAMPLE 8

Expression of P. multocida toxin in E. coli

The amount of PMT produced in different recombinant E. coli clones wasdetermined by estimating the incorporation of S-35 labelled methionineusing the following procedure. Cells were grown in AB minimal medium(ref. 21) supplemented with 0.2% glucose, 1 μg/ml thiamine and 50 μg/mlleucine at 37° C. At an optical density (OD₄₅₀) of 0.6, 1 μl of a 1mCi/ml S-35 methionine solution (purchased from Amersham, S J. 1015) wasadded to a 1 ml sample of the culture at 37° C. 3 minutes later, 50 μlof 10 mg/ml unlabelled methionine was added as a chase and after further3 minutes, the sample was put on ice. Samples were pelleted (6000×g for5 minutes) and resuspended in loading buffer (0.5M Tris-HCl pH 6.8, 3%SDS, 15% glycerol, 5% mercaptoethanol, bromophenol blue). Subsequently,proteins were separated in SDS-PAGE as described in Example 5.Furthermore, the gel was dried and subjected to autoradiographyovernight on a KODAK XAR-5 X-ray film. Relative amounts of PMT wereestimated by scanning of the X-ray film, using the β and β' subunits ofthe E. coli RNA-polymerase as a reference.

Recombinant clones tested in this way were MT102 strains harbouring theplasmids pSPE 312, pSPE 525 (shown in FIG. 11) and pSPE 481 (shown inFIG. 12), respectively. Plasmid pSPE 481 consists of the 7 kb PstIfragment of pSPE 525 ligated to a 1.3 kb PstI fragment of plasmid pPL195, containing part of the ampicillin resistance gene and the lambda PLpromoter and operator region. pPL 195 was constructed by inserting apUC8 EcoRI-HindIII polylinker (ref. 33) into a EcoRI and HindIIIrestricted PLc28 vector (ref. 34). The resulting pSPE 481 plasmidcarries the lambda PL promoter transcribing into the pmt gene.

                  TABLE 4                                                         ______________________________________                                        Estimated amount of PMT in the recombinant E. coli clones                     ______________________________________                                                      SPE312  SPE525     SPE481                                       ______________________________________                                        molecules/cell                                                                              <500    3000-4600  12000-15000                                  ______________________________________                                        Data used in the above calculations                                                         β  β'    PMT                                          ______________________________________                                        Methionine content (%)                                                                      2.76    2.56         2.80                                                     (23)    (24)                                                    Size (kD)      150     155       146.5                                                      (23)    (24)                                                    Molecules/cell                                                                              4500    4500                                                    (at 2.5-3 doublings/hour)                                                                   (25)    (25)                                                    ______________________________________                                    

Furthermore, the content of PMT in SPE481 was estimated using thefollowing procedure. A 100 ml culture with an optical density (OD₄₅₀) of5 was pelleted (6000×g for 5 minutes) and resuspended in 10 ml of 50 mMTris-HCl pH 7.0. The supernatant, which did not contain any PMT, wasdiscarded and the harvested cells were disrupted by sonication. PMT wasthen further purified on an anion-exchange column as described in ref.6. The purified PMT was then subjected to a quantitative ELISA asdescribed in Example 2, obtaining an estimated value of 2-5 μg PMT perml of culture fluid.

The Escherischia coli K-12 strain MT102 harbouring the pSPE 481 plasmidhas been deposited Mar. 21, 1988 according to the Budapest Treaty inDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under thestrain designation Escherichia coli K-12 SPE 481. The accession numberis DSM 4488.

EXAMPLE 9

Production and characterization of toxin derivatives

The following derivatives of toxin-encoding plasmids were constructedwith the purpose of producing truncated, i.e. detoxified, toxins whichare potentially useful for immunogenic purposes. The constructions wereprepared on the basis of the restriction mappings disclosed in Examples6 and 7. The hypothetical toxin-derived proteins produced from plasmidspSPE A through pSPE Q, proteins A through R, are shown in FIG. 13. Allderivatives were optimally expressed from the respective plasmids instrain SG 21059 kindly provided by Susan Gottesmann. The known genotypeof this strain is Δgal 1on146::ΔTn10 Δlac.

1) pSPE A. The plasmid was constructed by restricting pSPE 481 with therestriction enzyme StuI prior to ligation. This deletion causes a changeof reading frame. However, as described below, a PMT derivative could bedetected in Western blotting as well as in the EBL toxicity test. Thiscould be due to a small amount of erroneous frame-shifting in thetranslation procedure. See FIG. 13.

2) pSPE B. The plasmid was constructed by restricting pSPE 481 with therestriction enzyme XbaI prior to ligation. The plasmid codes for ahypothetical PMT derivative of about 108 kD, lacking amino acids 169through 468 of PMT. See FIG. 13.

3) pSPE C. The plasmid was constructed by restricting pSPE 481 with therestriction enzyme NdeI prior to ligation. This deletion causes a changeof reading frame. However, as described below, a PMT derivative could bedetected in small amounts. This could be due to erroneous frame-shiftingin the translation procedure. See FIG. 13.

4) pSPE D. The plasmid was constructed by restricting pSPE 481 with therestriction enzymes NdeI and SnaBI and subsequently blunt-ending theresulting ends, using T4 polymerase (purchased from New England Biolabs)as described by the manufacturer, prior to ligation. This deletioncauses a change of reading frame. However, as described below, a PMTderivative could be detected in small amounts. This could be due toerroneous frame-shifting in the translation procedure. See FIG. 13.

5) pSPE E. The plasmid was constructed by restricting pSPE 481 with therestriction enzyme HindIII prior to ligation. The plasmid codes for ahypothetical PMT derivative of about 53 kD, since this deletion causes achange of reading frame. See FIG. 13.

6) pSPE F. The plasmid was constructed by restricting pSPE 312 with therestriction enzyme HindIII prior to ligation. Like pSPE E, this plasmidcodes for a hypothetical PMT derivative of about 53 kD. See FIG. 13.

7) pSPE G. The plasmid was constructed by restricting pSPE 481 with therestriction enzymes SnaBI and XhoI and subsequently blunt-ending theresulting ends as above, prior to ligation. The plasmid codes for ahypothetical PMT derivative of about 135 kD, lacking amino acids 507through 568 of PMT. See FIG. 13.

8) pSPE H. The plasmid was constructed by restricting pSPE 481 with therestriction enzymes SnaBI and SpeI and subsequently blunt-ending theresulting ends as above, prior to ligation. The plasmid codes for ahypothetical PMT derivative of about 88 kD, lacking amino acids 569through 1058 of PMT. See FIG. 13.

9) pSPE I. The plasmid was constructed by restricting pSPE 481 with therestriction enzyme NsiI prior to ligation. The plasmid codes for ahypothetical PMT derivative of about 79 kD, lacking amino acids 634through 1204 of PMT. See FIG. 13.

10) pSPE J. The plasmid was constructed by restricting pSPE 312 with therestriction enzyme NsiI prior to ligation. The plasmid codes for ahypothetical PMT derivative of about 70 kD. See FIG. 13.

11) pSPE K. The plasmid was constructed by restricting pSPE 481 with therestriction enzyme SpeI and blunt-ending the resulting ends as aboveprior to ligation. The plasmid codes for a hypothetical PMT derivativeof about 117 kD. See FIG. 13.

12) pSPE L. The plasmid was constructed by restricting pSPE 312 with therestriction enzyme EcoRI prior to ligation. The plasmid codes for ahypothetical PMT derivative of about 124 kD. See FIG. 13.

13) pSPE O. The plasmid was constructed by partially restrictingnon-methylated pSPE 481 with the restriction enzyme BclI prior toligation. The plasmid codes for a hypothetical PMT derivative of 133 kD,lacking amino acids 30 through 150 of PMT. See FIG. 13.

14) pSPE P. The plasmid was constructed by restricting pSPE 481 with therestriction enzyme SpeI, subsequent treatment with the exonucleaseBal31, restriction with EcoRI and finally treatment with Klenow fragmentof DNA polymerase I in the presence of all four deoxyribonucleotidesprior to ligation. The plasmid codes for a hypothetical PMT derivativeof about 136 kD, lacking amino acids 1043 through 1130 of PMT. See FIG.13.

15) pSPE Q. The plasmid was constructed from a derivative of pSPE 481,pSPE 680. pSPE 680 was constructed by restricting pSPE 481 with therestriction enzymes BamHI and ClaI, and treatment with Klenow fragmentof DNA polymerase I in the presence of all four deoxynucleotides priorto ligation. Subsequently, pSPE Q was constructed by restricting pSPE680 with EcoRV prior to ligation. The plasmid codes for a hypotheticalPMT derivative of 127 kD, lacking amino acids 175 through 246 of PMT.

16) pSPE R. The plasmid was constructed by restricting pSPE 481 with therestriction enzymes SpeI and EcoRI and blunt-ending the resulting ends,as described above, prior to ligation. The resulting plasmid codes for ahypothetical PMT derivative of about 117 kD. See FIG. 13.

The reactivity of selected derivatives with a panel of anti-PMT MAbs hasbeen studied by sandwich ELISA's based on detection with non-competitivecombination pairs of MAbs. By this method an antigen-code (Ag-code),i.e. a term indicating the epitope-difference of the derivative comparedto PMT, was determined for each derivative.

The minimal cytopathic dose (MCD) on 120 μl of 1.5×10⁵ embryonic bovinelung cells has been estimated for some affinity-purified PMTderivatives.

The results of the two studies are indicated in the following table(Table 5):

                  TABLE 5                                                         ______________________________________                                        Derivative                                                                           L        O       P      B*    C*     G*                                Ag-code                                                                              PMT-α                                                                            PMT-β                                                                            PMT-β                                                                           PMT-γ                                                                         PMT-γ                                                                          PMT                               MCD (ng)                                                                             250      30000   5      n.d.  n.d.   n.d.                              ______________________________________                                         *not affinitypurified (sonicates)                                             n.d.: not done                                                           

The antigen-code "PMT" indicates that the derivative reactsindifferently compared to PMT by the sandwich-ELISA, i.e. all epitopescharacterized by the panel of MAbs, which are present on PMT can bedetected on the derivative. The Ag-code "PMT-x", where x is α, β or γindicates that the MAb(s) reacting with epitope x on PMT are notreacting with the derivative. The results of these tests shows thatepitope α is missing on derivative L, β is missing on O and P and γ ismissing on B and C.

The MCD of PMT is approx. 0.01-0.03 ng and it is therefore obvious thatthe abovementioned derivatives L and O are practically non-cytopathiccompared to PMT, whereas P shows slightly remaining cytopathic activity.

A Western blot was made using a mouse anti-PMT antiserum produced asdescribed in Example 1, and using horseradish perioxidase rabbitanti-mouse Ig whole antibody (purchased from Amersham) as the secondaryantibody. Strains harbouring the plasmids pSPE A, B, C, D, E, G, H, Iand L were shown to react with the anti-PMT antiserum (cf. FIG. 14).

Trials carried out on mice for elucidation of the immunogenic effect ofthe (O-derivative)

Purpose

To study the immunogenic effect of PMT after deletion in the N-terminalend (O-derivative).

Method

For the trials, BALB/c mice were used. Sexually mature female mice wereimmunized subcutaneously 2 times at 14 days interval with 0.3 mlO-derivative (2.5-5 μg/ml) in 20% _(A) lhydrogel (cf. Example 12) inPBS+0.1% zero-mouse-serum.

Simultaneously with the 1st vaccination the female mice were mated.Thus, the 2nd vaccination took place about 1 week before expected birth.

1) About 10 days old the baby mice were divided into 2 groups. Half ofthem were dripped intranasally with PMT (totally 60 ng PMT), the otherhalf was injected intraperitoneally (i.p.) with PMT in differentconcentrations. The number of dead animals was recorded.

2) All the adult female mice were bled after killing of the survivingbaby mice. The blood samples were analysed by ELISA as described inExample 13 for the presence of antibodies against PMT. Immediately afterblood sampling the mice were injected i.p. with different concentrationsof PMT. The number of dead animals was recorded.

    ______________________________________                                        Schematic trial plan                                                          Trial                                                                         week                                                                          No.                                                                           ______________________________________                                        3    31 female BALB/c mice were mated and subsequently immunized                   with O-derivative                                                        1    31 female BALB/c mice were immunized for the second time                      with O-derivative                                                        0    31 female mice delivered 122 baby mice                                   1    the baby mice were divided into group I (61) which was                        treated intranasally with PMT. Group II (61) which was                        treated i.p. with PMT                                                    2    surviving baby mice were killed                                               female BALB/c mice were bled                                                  female BALB/c mice were injected i.p. with PMT                           3    the number of surviving female BALB/c mice was recorded and                   the animals killed                                                            Trial finished                                                           ______________________________________                                    

Results are shown in tables 6 and 7.

LD₇₅ in non-protected baby mice was about 20 ng PMT injected i.p.

LD₅₀ in non-protected adult mice was about 70 ng PMT injected i.p.

Conclusion

It can be concluded that mice born from mothers vaccinated withO-derivative vaccine in the doses described can survive i.p. injectionof min. 25×LD₅₀ of PMT. The protection is obtained via antibodiestransferred via colostrum from mother to offspring.

Furthermore it can be concluded that O-derivative vaccinated animalsdevelop antibodies against PMT, even if some variation is seen. The micecan survive i.p. injection of min. 50×LD₅₀ of PMT.

Thus, mice vaccinated with O-derivative can transfer a considerableprotection against PMT to the offspring via colostrum. The micethemselves develop antibodies against PMT and are protected against evenhigh concentrations of PMT.

                  TABLE 6                                                         ______________________________________                                        SCHEME OF VACCINATION TRIALS WITH O-FRAGMENT                                  IN MICE (ADULT)                                                               CO-                                                                           NC.        Amount of PMT (challenge)                                          Tr- x, (s)  (vac-  200  250  800  3200 4000 8000 16000                        ial (ln titer)                                                                            cine)  ng   ng   ng   ng   ng   ng   ng                           ______________________________________                                        1   6.49    5      0/2*      0/4  0/4                                             (0.60)  μg/ml                                                          2   1.27    5                          1/3  3/4  4/4                              (1.06)  μg/ml                                                          3   1.06    2.5         1/2            2/4       3/4                              (1.10)  μg/ml                                                          ______________________________________                                         *Number of dead mice/number of i.p. injected mice                        

                  TABLE 7                                                         ______________________________________                                        SCHEME OF TRIAL WITH O-FRAGMENT IN MICE (OFFSPRING)                           Amount of PMT (challenge)                                                     Trial 20 ng   30 ng   100 ng 150 ng 500 ng 750 ng                             ______________________________________                                        1     0/4*            0/7           0/7                                       2             0/9            0/10          8/8                                3             0/1            0/10          4/5                                ______________________________________                                         *Number of dead mice/number of i.p. injected mice                        

EXAMPLE 10

Differentiation of PMT⁺ and PMT⁻ strains by PMT-ELISA

615 field isolates and 7 reference strains of P. multocida wereexamined. The field isolates were obtained from nasal swabs (603isolates) and lungs (12 isolates) of pigs from 156 Danish herds and wereidentified by the following criteria: acid produced from glucose,saccharose, mannitol, sorbitol and not from maltose, arabinose, dulcitoland inositol; and production of indole, ornithine decarboxylase,catalase, oxidase and not of urease.

Extracts for toxin analyses were prepared by harvesting blood agar (9 cmPetri dish) overnight (37° C.) cultures into 2 ml of sterile water bymeans of a spatula. The suspensions were left for extraction. at 37° C.for approximately 18 hours. One part of the extract was examineddirectly by PMT-ELISA as described in Example 2. All absorbances (A)were expressed as percentages of the absorbance obtained by a positivecontrol (A_(O)). This control was a 1:1 dilution of an extract, freshlyprepared for each test of the toxigenic type D reference strain P.multocida ssp. multocida 45/78.

Another part was centrifuged (30 min. at 1500×g), the supernatantsterile filtered and subsequently examined in the EBL-cell test asdescribed earlier refs. 22 and 29).

The 615 field isolates were characterized as toxigenic (250) ornon-toxigenic (365) by the EBL-cell test and were of capsular type A(119 toxigenic and 92 non-toxigenic isolates) or D (131 toxigenic and273 non-toxigenic isolates).

Full agreement between the EBL-cell test and the PMT-ELISA was obtainedfor the 615 field isolates and the 7 reference strains (Table 8).

                  TABLE 8                                                         ______________________________________                                                         EBL-cell-test                                                                         PMT-ELISA                                                             a)      b)                                                   ______________________________________                                        250 field isolates of                                                                            +         +                                                P. multocida ssp. multocida                                                   365 field isolates of                                                                            -         -                                                P. multocida ssp. multocida                                                   Type strain (CCUG 17977)                                                                         -         -                                                P. multocida ssp. septica                                                     Type strain (NCTC. 10204)                                                                        -         -                                                P. multocida ssp. galicida                                                    Type strain (NCTC 10322)                                                                         -         -                                                P. multocida ssp. multocida, type A                                           Reference strain (ATCC 12945)                                                                    -         -                                                P. multocida ssp. multocida, type A                                           Reference strain (NCTC 12177)                                                                    +         +                                                P. multocida ssp. multocida, type A                                           Reference strain (ATCC 7707)                                                                     -         -                                                P. multocida ssp. multocida, type D                                           Reference strain (NCTC 12178)                                                                    +         +                                                P. multocida ssp. multocida, type D                                           ______________________________________                                         a) All EBLpositive (+) bacterial extracts bad EBLtitres above 10.sup.3        (median 10.sup.4, range 10.sup.3 -10.sup.6), in the EBLcell test,             EBLnegative (-) extracts were noncytopathic.                                  b) All 1:1 diluted ELISApositive (+) bacterial extracts had relative          absorbances above 39% (mean ± SD: 94% ± 13%) in the PMTELISA,           whereas all ELISAnegative () extracts had relative absorbances below 9%       (2.1% ± 1.9%).                                                        

The cytopathic and non-cytopathic extracts of the 615 field isolateswere separated in two clearly distinguishable groups by the PMT-ELISA(FIG. 15). Since the mean±SD of the absorbances (A) obtained from the1:1 diluted extracts of the 250 toxigenic isolates was 1.72±0.48, visualreadings instead of photometric measurements of the ELISA-results wouldbe satisfactory for the differentiation of extracts of P. multocida. Themean±SD of the PMT-concentration in the extracts of the toxigenicisolates of P. multocida was estimated being 2.8±1.9 μg/ml, and sincethe detection limit of the PMT-ELISA is approx. 50 pg (1 ng/ml) PMT (cf.Example 2), dilutions of the extracts (FIG. 16) and extracts with lowPMT-concentrations may appropriately be tested by PMT-ELISA. The mainadvantages of the PMT-ELISA compared to existing tests are theindependence of cell culture or laboratory animal facilities, theability of a single laboratory worker to handle several hundreds ofsamples per day and the possibility of obtaining quantitative objectiveresults from bacterial extracts in 4 hours.

EXAMPLE 11

Neutralization of PMT with monoclonal anti-PMT-antibodies

Samples (30 μl) of either PMT in PBS or PMT in a crude cell-free extractof P. multocida 45/78 (ref. 6) containing PMT in amounts up to 12 ng and1 μg of purified MAb (P3F51) were incubated for 15 min. at 20° C. beforeapplication to embryonic bovine lung (EBL) cells (120 μl, 1.5×10⁵cells/ml) as described for the original EBL-cell test (ref. 29). Theminimal cytopathic dose (MCD) of PMT was estimated when no MAb waspresent in the sample. The neutralization titer was recorded as thenumber of MCD which could be neutralized by 1 μg of MAb.

The results appear from Table 9 below.

                  TABLE 9                                                         ______________________________________                                        Hybridoma   Representa-                                                                             Neutralization in                                       group No.   tive MAb  EBL-cell test (x MCD).sup.a                             ______________________________________                                        1           P3F51     130                                                     2           P3F64     70                                                      3           P3F37     <2                                                      4           P4F58     30                                                      5           P3F22     40                                                      6           P4F46     100                                                     7           P4F38     35                                                      8           P4F55     40                                                      9           P3F50     400                                                     10          P3F53     55                                                      ______________________________________                                         .sup.a) Neutralization of the cytopathic effect on PMT was estimated as       the number of minimal cytopathic doses (MCD) neutralized by 1 μg of        MAb. The MCD of PMT is about 30 pg.                                      

As indicated in Table 9, addition of 1 μg of MAb to PMT 15 min. beforeaddition to the EBL-cells resulted in a 30 to 400 times increase of theMCD for 9 out of the 10 representative MAbs, whereas MAb P3F37 had avery low neutralizing effect on PMT. The neutralization of thecytopathic effect of PMT was also achieved when a crude cell-freeextract of P. multocida 45/78 was used instead of pure PMT.

Samples (200 μ) containing PMT in variable amounts up to 2.56 μg andpurified MAb (P3F51) in amounts between 0.15 and 15 μg were incubatedfor 15 min. at 20° C. and injected intraperitoneally (i.p.) in femaleBALB/c mice (6 weeks old, 15 to 20 g). Mice dying within a week from thetime of PMT injection were recorded and the lethal dose of PMT and theneutralizing effect of the MAb was estimated. When 1.5 or 15 μg of P3F51were added the lethal dose of PMT increased about 4 and 32 times,respectively, whereas 0.15 μg of the MAb had no neutralizing effect.

To study the in vivo neutralization ability of anti-PMT monoclonalantibodies a 200 μl solution containing 15 μg of purified monoclonalantibody (P3F51) was injected (i.p.) in female BALB/c mice (6 weeks old,15-20 g) 2 days before i.p. administration of a 200 μl solutioncontaining PMT in varying amounts up to 2.56 μg either in a pure form oras a crude cell-free extract of P. multocida 45/78 (ref. 6). Theneutralizing effect was estimated as described above.

The lethal dose of PMT increased about 32 times when mice were passivelyimmunized with 15 μg of P3F51 2 days before challenge with PMT or acrude cell-free extract of P. multocida 45/78.

EXAMPLE 12

Vaccination with purified PMT or derivative O

15 mg of PMT purified as described in Example 3 in 45 ml of PBS wasdialyzed against 0.35% formaldehyde in PBS, pH 7.3-7.9, for 36 hours at4° C. after which 1 g/l lysine-HCl was added to the dialysis liquid, andafter 18 hours the dialysis was continued with repeated changes of PBS.The thus produced detoxified PMT was analyzed for lack of (orsufficiently reduced) toxic activity in the mouse lethality test and thecytopathic test on EBL-cells described above as well as a dermonecrotictest in guinea pigs as described by Foged et al. (1).

10 mg of biologically inactive (detoxified) PMT in 40 ml PBS was thencoupled to 10 ml aluminium hydroxide gel purchased from Superfos,Denmark, under the trade name Alhydrogel as recommended by themanufacturer and diluted in 20% aluminium hydroxide in PBS to a finalconcentration of about 5 μg/ml or 125 μg/ml detoxified PMT.

Gestating gilts were immunized subcutaneously 4-6 weeks and 2-3 weeksbefore farrowing with a dosis of 3 ml of the detoxified PMT vaccinecomposition prepared above. After farrowing piglets were inoculatedintranasally with Bordetella bronchiseptica and P. multocida asdescribed in ref. 1 and the protective effect of the immunization of thesows was estimated by measuring the average daily weight gain beforeslaughtering of the pigs (at about 90 kg live weight) and determinationof osteopathological conditions in the snout of the pigs atslaughtering. Pigs from immunized sows were compared to pigs fromnon-immunized sows and the protective effect of the immunization isshown in Table 10.

                  TABLE 10                                                        ______________________________________                                                        Mean daily  No. of animals                                           No. of animals                                                                         weight gain with severe tur-                                         (litters)                                                                              after weaning                                                                             binate atrophy (%)                                ______________________________________                                        Pigs from                                                                              61 (8)     781 g       49 (80.3%)                                    non-immunized                                                                 sows                                                                          Pigs from                                                                              174 (20)   848 g       20 (11.5%)                                    immunized                                                                     sows                                                                          ______________________________________                                    

In a study still in progress gilts were immunized with 50 μg/dose ofaffinity-purified derivative O from sonicates of an E.coli clonecontaining pSPE O as described in Example 9. No modifications of O wereperformed except for coupling to Alhydrogel. Preliminary results of thevaccination study indicates that:

a) the serum- and colostrum titers against native PMT are similar forgilts vaccinated with derivate O and formaldehyde treated PMT,

b) the specific antibodies are transferred to piglets through colostrumequally well in both vaccine groups.

c) the clinical symptoms of atrophic rhinitis are prevented equally wellin the progeny from gilts vaccinated with O (O-piglets) and formaldehydetreated PMT (P-piglets), and that this prevention seems to be close to100%, when compared to piglets born by unvaccinated gilts (controlpiglets).

d) the toxigenic P. multocida used for the experimental infection can bereisolated in significantly higher rates from control piglets than fromO- or P-piglets at 5 weeks of age.

EXAMPLE 13

Detection of anti-PMT-antibodies

By proceeding substantially as described in Example 2, but by incubatingthe coating monoclonal anti-PMT-antibody with a premixed preparation ofserum and a constant amount of PMT, it is possible to detectanti-PMT-antibodies in serum of for instance pigs infected with P.multocida or of animals vaccinated with a vaccine of the invention. Themixture which was prepared for concentrated or diluted serum samples wasincubated for 30 min. at 37° C. before incubation for 15 min. in themicrotiter plate. The presence of anti-PMT-antibodies in the serumsample was detected by a decrease in absorbance measured substantiallyas described in Example 1 (the section entitled "ELISA for estimatingepitope specificity"). The results are shown in FIG. 17 which shows the50% blocking titers of serum from an anti-PMT-antibody negative pig(<2), a pig infected with a toxin-producing P. multocida strain (about14) and a gilt vaccinated with the vaccine described in Example 12(about 250).

EXAMPLE 14

Detection of PMT by colony blot and immunoblotting

The presence of PMT in samples may be detected by a colony blot method(ref. 14) as described in Example 5 (the section entitled "Screeningprocedure")

Similarly, the presence of PMT in samples may be detected by separatingproteins in the samples electrophoretically by SDS-PAGE (as described inExample 1) and transferring them electrophoretically to a nitrocellulosemembrane where PMT, if present, can be visualized by immunoblotting asdescribed in Example 1 (in the section entitled "Immunoblotting"). Theelectrophoretic location of the stained protein band also gives theapparent molecular weights of PMT (approximately 143 kd).

EXAMPLE 15

Genetic distinction between PMT+ and PMT- Isolates of P. multocida asdetermined by colony hybridization

P. multocida isolates (17 toxin-positive and 18 toxin-negative strainsas determined by ELISA and EBL tests as described above) were inoculatedon Tryptic Soy Broth Agar plates (purchased from DIFCO). Afterincubation overnight at 37° C., a replica was made on a nitrocellulosemembrane filter (Schleicher & Schull BA 85). This replica was placed(face up) on top of 4 consecutive Whatman 3MM filters which were soakedin 10% SDS, denaturation buffer (0.5M NaOH, 1.5M NaCl), neutralizationbuffer (0.5M Tris-HCl pH 8.0, 1.5M NaCl) and 2×SSPE (360 mM NaCl, 20 mMNaH₂ PO₄, 2 mM EDTA pH 7.4), respectively. Incubation was carried outfor 5 minutes on each filter at room temperature. Subsequently, thenitrocellulose filter was dried and DNA was fixed to the filter bybaking at 80° C. for 2 hours. Prehybridization and hybridizations weredone in 6×SSC (0.15M NaCl, 0.015M sodium citrate, pH 7), 0.5% SDS and5×Denhardt solution for 2 hours and overnight, respectively, at 65° C.The probe was a radioactively labelled XbaI fragment from position 1623to 4376 in the sequence shown in FIGS. 10 (a)-(j) (SEQ ID NO:1) preparedby the nick translation method (ref. 22). After hybridization the filterwas washed at 25° C. in 2×SSC, 0.5% SDS for 2×15 min. and in 0.2×SSC,0.5% SDS for 2×1 hour at 65° C. and left for autoradiography overnight.

The results appear from FIG. 18 which shows that colonies at positions5, 6, 8, 9, 10, 12, 13, 14, 15, 17, 19, 22, 34, 36, 37, 39, 45 and 50were PMT⁺ and colonies at positions 7, 23, 24, 26, 28, 30, 32, 42, 44,47, 48, 53, 56, 58, 63, 66, 73 and 75 were PMT⁻. These results are inaccordance with the ELISA and EBL determinations. Hence, non-toxigenicstrains of P. multocida owe their lack of toxin production to a lack ofthe PMT encoding pmt gene.

EXAMPLE 16

Purification of rPMT and comparison of rPMT with PMT

In the toxin purification procedure, cells harvested from a 1 lovernight stationary culture of SPE312 were resuspended in 10 ml of H₂ Oand sonicated several times for 0.5 min. at 0° C. using a Bransonsonifier 250 (Branson, Conn., U.S.A.). The sonicate was diluted to 50 mlin 0.1 M Tris-HCl, pH 7.8 containing 0.5 M NaCl before appliation to theaffinity column which was prepared by immobilizing the anti-PMT MabP3F51 as described in Example 3. After repeated washings of the affinitycolumn, rPMT was eluted with 0.1 M glycin-HCl, pH 2.8 as earlierdescribed for the affinity purification of PMT from extracts oftoxigenic P. multocida. All fractions were immediately neutralized with1 M K₂ HPO₄.

A diluted bacterial sonicate of SPE312 containing approximately 82 μg ofrPMT as determined by the quantitative ELISA described in Example 2 wasapplied to a 1-ml affinity column to which was coupled approximately 5mg of anti-PMT MAb P3F51. No rPMT could be detected in the effluent fromthe column. Upon elution approximately 75 μg of rPMT was obtained in thetwo main fractions of 1.4 ml each. This corresponds to a recovery of 91%of the applied rPMT.

PMT assays

Quantification of rPMT was done as described for PMT (Example 2) usingthe capture anti-PMT MAb P3F51 and the biotinylated detector MAb P3F37in the PMT-ELISA, a sandwich ELISA based on the same technique asexplained below for the study of epitopes on rPMT and PMT.Quantification by the PMT-ELISA was compared to results obtained in amodified Coomassie brilliant blue dye-binding microassay previously usedfor the determinations of protein concentrations and dye-binding abilityof PMT compared to bovine serum albumine (BSA).

Comparison of epitopes on rPMT and PMT was done by sandwich ELISA'sbased on 10 anti-PMT MAbs (Example 1) purified from hybridomasupernatants on protein A-agarose columns. These MAbs have been shown toreact with different epitopes on PMT. The sandwich-ELISA's were done asdescribed in Example 2. Dual determinations were performed for bothantigens in all 100 combinations of the 10 catching MAbs and the samebiotinylated detecting MAbs. Combination pairs of MAbs resulting inabsorbances below 0.3 were considered competitive. For thenon-competitive combination pairs, the results were described as themean of dual determinations of absorbance obtained for rPMT relative tothe mean of dual determinations of absorbance for PMT.

The dermonecrotic and lethal effects of rPMT and PMT were determined byinjecting 200 μl of dilutions of the previously ELISA-quantified samplesintradermally into guinea pigs or intraperitoneally into BALB/c mice,respectively. Samples resulting in a dermal lesion of 10 mm or more at48 h after intradermal injection were scored as dermonecrotic andsamples resulting in death in less than 5 days after intraperitonealinjection were scored as lethal. All results were based on at leastduplicate determinations.

Affinity-purified rPMT and PMT had very similar patterns of reactions inthe structural ELISA test based on 100 combination pairs of 10 differentanti-PMT MAbs and 100 ng/ml of affinity purified antigen. For PMT 25pairs resulted in an absorbance value (A₄₉₂) below 0.3 which wasconsidered to indicate competitiveness. The same 25 pairs showedcompetitive reactions when the antigen was 100 ng/ml rPMT. The remaining75 non-competitive combination pairs resulted in A₄₉₂ values above 0.3both when PMT and rPMT was used. The overall mean±SD for the 75calculated values of the relative absorbances of rPMT compared to PMTwas 112%±8%. Only minor differences from the overall mean were observedfor the mean values for the 10 catching MAbs and the 10 biotinylateddetector MAbs.

PMT and rPMT reacted very similarly when tested for cytopathic effect onEBL-cells, for dermonecrotic activity in guinea pigs, and for lethalityin mice, and their ability to bind Coomassie brilliant blue were equaland approximately 2.5 times weaker than the dye-binding ability of BSA(Table 11).

                  TABLE 11                                                        ______________________________________                                        Cytopathic,                                                                   dermonecrotic, lethal and dye-binding effects of PMT and rPMT                       minimal   minimal     minimal                                                 cytopathic                                                                              dermonecrotic                                                                             lethal  dye-binding                               Sample                                                                              dose (pg) dose (ng)   dose (ng)                                                                             (%).sup.a                                 ______________________________________                                        PMT   20-40     15-45       25-50   40-45                                     rPMT  20-40     35          30      35-45                                     ______________________________________                                         .sup.a The concentration of BSA relative to the concentration of sample       resulting in equal colour formation in the Coomassie brilliant blue           dyebinding microassay.                                                   

EXAMPLE 17

Examination of E.coli and P. multocida sonicates for cytopathic activity

Sonicates of E.coli SPE312 and P. multocida 45/78, prepared as describedin Example 16 were tested for cytopathic effect in the embryonic bovinelung (EBL) cell test (ref. 29). A row of 5-fold dilutions was preparedfor each sonicate and 30 μl of each sample was applied to 1.8×10⁴EBL-cells in 120 μl of culture medium and the mixture incubated for 3days at 37° C. before fixation and staining. Samples which resulted inmonolayers of EBL-cells morphologically discernible from theepithelial-like swirling patterns of negative control culture, werescored as cytopathic. The cytopathic effects for affinity purified rPMTand PMT in the EBL-cell test were determined in the same way. Theminimal cytopathic dose (MCD) for the samples was calculated as minimalamount of rPMT or PMT, determined by the quantitative PMT-ELISA causinga cytopathic effect.

Neutralization of the cytopathic effect of E.coli SPE 313 sonicate byanti-PMT MAbs was compared to neutralization of pure PMT: Samples (30μl) containing approximately 1 μg of MAb and varying amounts of sonicateor PMT were incubated for 15 min. at 20° C. before application toEBL-cells. The results were recorded as the number of MCDs neutralizedby each MAb, and as the ratio between the number of neutralized MCDs ofthe sonicate and pure PMT for each MAb.

Sonicates of SPE308 and SPE312 were shown to cause morphological changesof embryonic bovine lung (EBL) cells, identical to those caused bytoxigenic strains of P. multocida (FIG. 19 (data for SPE308 not shown)).As observed for pure PMT, the cytopathic effect of the sonicate ofE.coli SPE312 could be neutralized by incubation with anti-PMT MAbs.Between 5 and 125 times MCD of the sonicate could be neutralized byvarious anti-PMT MAbs, whereas between 3 and 125 times MCD of the purePMT were neutralized. The overall mean±SD for the 10 calculated valuesof the relative number of neutralized MCDs of E.coli SPE312 sonicatecompared to PMT was 95%±32X. A PMT-unrelated MAb used as a control didnot neutralize the effects of the two cytopathic preparations.

EXAMPLE 18

Analysis of the nature of the DNA flanking the pmt gene

In an attempt to investigate the nature of the DNA flanking the pmt genein P. multocida 45/78, chromosome walking was performed as described inref. 37. By using a colony hybridization procedure plasmids carrying P.multocida DNA were isolated from the P. multocida gene library describedin Example 4.

Probes

The plasmid pLOL03 was constructed by subcloning a 0.8 kb AccI-HindIIIDNA fragment of pSPE344 (FIG. 20) in the vector pGEM-blue (Promega,Wis., USA). The plasmid pLOR02 was likewise constructed by subcloningthe 2.4 kb EcoRI-BglII fragment of pSPE312 (FIG. 20) in the vectorpGEM-blue. The E.coli K12 strain DH5alpha (BRL, Md., USA) was used ashost strain for pLOL03 and pLOR02. pLOL03 and pLOR02 in linearized formswere used for generating RNA probes of the P. multocida DNA carried bythese plasmids. The RNA probes were radioactively labelled using theRiboprobe System II procedure (Promega, Wis., USA), and used in thecolony hybridizations and Southern blots described below.

Colony hybridization

The P. multocida gene library was spread in appropriate dilution onseveral LB-plates containing 10 μg/ml tetracycline, and incubatedovernight at 37° C. Replicas of the plates were made on nitrocellulosemembrane filters, and the cells were lysed and the DNA fixed to thefilters as described in example 15.

Prehybridization and hybridization was performed at 65° C. in 50%formamide, 6×SSC (0.15 NaCl, 0.015 M tri-sodium citrate, pH 7.0), 0.1%SDS, 5×Denhardt's solution and 200 μg/ml denatured Salmon sperm DNA forat least 2 hours and overnight, respectively. After hybridization, thefilters were washed twice at room-temperature in 1×SSC, 0.1% SDS, andtwice at 65° C. in 0.1×SSC, 0.1% SDS. After washing, the filters wereleft overnight for autoradiography.

This procedure resulted in the isolation of a number of clones carryingP. multocida DNA flanking the inserts in pSPE308 or pSPE312. Theseclones were further analyzed using the Southern blot technique (ref.17). The Southern blots showed that the following plasmids wererecognized by the RNA probe coded for by pLOL03: pLOA01, pLOA02 andpLOA03. Similarly the plasmids pLOB01, pLOB02 and pLOB03 were recognizedby the RNA probe coded for by pLOR02.

pLOA03 (approx. 14.2 kb) and pLOB03 (approx. 12.7 kb) carried thelargest inserts. Their restriction maps and a Southern blot analysisshow that pLOA03 and pSPE308 contain overlapping DNA of approximately4.0 kb and that pLOB03 and pSPE312 contain overlapping DNA ofapproximately 1.7 kb as shown in FIG. 20.

A Southern blot was made using DNA extracted as described in Example 4(a KI gradient (0.875 g/ml) was used instead of the CsCl₂ gradient) fromthe toxigenic P. multocida 45/78 and from a non-toxigenic P. multocidastrain MH81P8, type D (ref. 36) and the plasmids pLOA03, pLOA02,pSPE308, pSPE312 and pLOB03 digested by restriction enzymes as indicatedin FIG. 21. The probe was the 2.4 kb BglII-EcoRI fragment of pLOB03radioactively labelled by nicktranslation (Rigby et al., 1977, (ref.19)). The result shows that:

1) The probe recognizes a DNA sequence on each of the plasmids pLOA03and pLOB03. Thus, there is a homologous sequence on each side of the pmtgene. The distance between these homologous sequences is approximately25 kb.

2) The probe recognizes distinct fragments of the chromosomal DNA ofboth P. multocida strains used in this Southern blot.

The above findings could indicate that the DNA flanking the pmt gene andthus the pmt gene itself has originally been carried by a bacteriophage,a transposon, a plasmid or another genetic element which is integratedinto the bacterial chromosome.

Dot blot

DNA from 24 bacteriophages isolated from P. multocida strains and allshown to be different in their lysis patterns towards a range of P.multocida strains were bound to a nylon filter by dot blotting. Theplasmid pLOA03 was radioactively labelled by nicktranslation, and usedas a probe against the filter. Hybridization and washing conditions wereas described above. The results are shown in FIG. 22. The probehybridized to 22 out of 24 bacteriophages and, as expected, to the fourpositive controls. By using pSPE308 and pLOB03 as probes, similarresults were obtained. pSPE312 gave only a slight hybridzation to someof the bacteriophage genomes. The 4.5 kb pmt gene containing ClaI-PvuIIfragment of pSPE312 (FIG. 5) did not show any homology to any of thebacteriophage genomes (autoradiographs are not shown).

These results show that there are sequences homologous to P. multocidabacteriophage DNA on both sides of the pmt gene. This furthersubstantiates the notion that the pmt gene is carried by a prophage.

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37. K. Kaiser and N. Murry, 1985: The use of phage lambda replacementvectors in the construction of representative genomic DNA libraries. In:DNA cloning, Vol. I, A practical approach, D. M. Glover (ed.) IRL Press,Oxford.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 2                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 4380 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 219..4076                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - AACAAGGGAA AATAGCTAGA TTAGACGATA TCGATAATAT CATAAATAAT AT - #TTAAAAAT         60                                                                          - TACGCCCCTT GACCTAGAGG GGCTTTTTTA TTACATCAAA AAAATAAACC CA - #AACACTGC        120                                                                          - GAATGTTTGG GGTTTTATTT ATAACCAAAA TACATTAATA TGTTTATTAA GT - #AAGCATTA        180                                                                          #AAA CAT        233AACT AACATAGAGG TTATGGAT ATG AAA ACA                       #      Met Lys Thr Lys His                                                    #     5  1                                                                    - TTT TTT AAC TCA GAT TTT ACT GTA AAA GGA AA - #A AGT GCC GAT GAA ATT          281                                                                          Phe Phe Asn Ser Asp Phe Thr Val Lys Gly Ly - #s Ser Ala Asp Glu Ile           #                 20                                                          - TTT AGA AGA TTG TGT ACT GAT CAT CCT GAC AA - #G CAA TTA AAC AAT GTA          329                                                                          Phe Arg Arg Leu Cys Thr Asp His Pro Asp Ly - #s Gln Leu Asn Asn Val           #             35                                                              - AAA TGG AAA GAA GTT TTT ATT AAT CGT TTT GG - #T CAG ATG ATG CTA GAT          377                                                                          Lys Trp Lys Glu Val Phe Ile Asn Arg Phe Gl - #y Gln Met Met Leu Asp           #         50                                                                  - ACT CCT AAT CCG AGA AAG ATT GTA GAA AAA AT - #T ATT AAT GAA GGG CTT          425                                                                          Thr Pro Asn Pro Arg Lys Ile Val Glu Lys Il - #e Ile Asn Glu Gly Leu           #     65                                                                      - GAA AAA CAA GGC CTG AAA AAT ATA GAT CCT GA - #A ACT ACA TAT TTC AAC          473                                                                          Glu Lys Gln Gly Leu Lys Asn Ile Asp Pro Gl - #u Thr Thr Tyr Phe Asn           # 85                                                                          - ATT TTT TCA TCT TCT GAC AGC TCC GAT GGG AA - #C GTT TTT CAT TAT AAC          521                                                                          Ile Phe Ser Ser Ser Asp Ser Ser Asp Gly As - #n Val Phe His Tyr Asn           #                100                                                          - TCT TTA TCA GAA TCC TAT CGA GTT ACT GAT GC - #C TGC CTA ATG AAT ATT          569                                                                          Ser Leu Ser Glu Ser Tyr Arg Val Thr Asp Al - #a Cys Leu Met Asn Ile           #           115                                                               - TTT GTG GAG CGT TAT TTT GAT GAT TGG GAC TT - #G CTA AAT AGC TTA GCC          617                                                                          Phe Val Glu Arg Tyr Phe Asp Asp Trp Asp Le - #u Leu Asn Ser Leu Ala           #       130                                                                   - AGT AAT GGA ATA TAT TCA GTA GGA AAA GAA GG - #A GCT TAT TAT CCT GAT          665                                                                          Ser Asn Gly Ile Tyr Ser Val Gly Lys Glu Gl - #y Ala Tyr Tyr Pro Asp           #   145                                                                       - CAT GAT TAT GGT CCA GAA TAT AAC CCT GTT TG - #G GGA CCA AAC GAA CAA          713                                                                          His Asp Tyr Gly Pro Glu Tyr Asn Pro Val Tr - #p Gly Pro Asn Glu Gln           150                 1 - #55                 1 - #60                 1 -       #65                                                                           - ATT TAC CAT TCT AGA GTG ATT GCA GAT ATC CT - #T TAT GCT CGC TCC GTA          761                                                                          Ile Tyr His Ser Arg Val Ile Ala Asp Ile Le - #u Tyr Ala Arg Ser Val           #               180                                                           - TGG GAT GAA TTT AAA AAA TAC TTC ATG GAG TA - #T TGG CAA AAA TAT GCT          809                                                                          Trp Asp Glu Phe Lys Lys Tyr Phe Met Glu Ty - #r Trp Gln Lys Tyr Ala           #           195                                                               - CAG CTT TAT ACC GAA ATG TTA TCT GAT ACA TT - #T CTT GCA ATG GCT ATT          857                                                                          Gln Leu Tyr Thr Glu Met Leu Ser Asp Thr Ph - #e Leu Ala Met Ala Ile           #       210                                                                   - CAG CAA TAT ACA CGA CAA ACG CTT ACT GAT GA - #A GGC TTT CTT ATG GTT          905                                                                          Gln Gln Tyr Thr Arg Gln Thr Leu Thr Asp Gl - #u Gly Phe Leu Met Val           #   225                                                                       - TGT AAC ACA TAT TAT GGC AAT AAG GAA GAA GT - #T CAA ATA ACT CTA CTA          953                                                                          Cys Asn Thr Tyr Tyr Gly Asn Lys Glu Glu Va - #l Gln Ile Thr Leu Leu           230                 2 - #35                 2 - #40                 2 -       #45                                                                           - GAT ATC TAT GGA TAC CCT TCC ACT GAT ATA AT - #T TGT ATA GAG CAA AAA         1001                                                                          Asp Ile Tyr Gly Tyr Pro Ser Thr Asp Ile Il - #e Cys Ile Glu Gln Lys           #               260                                                           - GGG CTT CCT ACT CCT AAA GTG ATA CTT TAC AT - #T CCT GGA GGA ACA CAA         1049                                                                          Gly Leu Pro Thr Pro Lys Val Ile Leu Tyr Il - #e Pro Gly Gly Thr Gln           #           275                                                               - CCA TTT GTT GAA TTT CTT AAT ACA GAT GAT CT - #G AAA CAA TGG ATT GCA         1097                                                                          Pro Phe Val Glu Phe Leu Asn Thr Asp Asp Le - #u Lys Gln Trp Ile Ala           #       290                                                                   - TGG CAT TTA AAA GAT AAC AAA CAT ATG GTC CG - #A TTC CGC AAA CAT TTC         1145                                                                          Trp His Leu Lys Asp Asn Lys His Met Val Ar - #g Phe Arg Lys His Phe           #   305                                                                       - TCG CTA AAA CAA CGT CAG GAA GGA GAA ACG TT - #T ACA GGT ATA GAT AAA         1193                                                                          Ser Leu Lys Gln Arg Gln Glu Gly Glu Thr Ph - #e Thr Gly Ile Asp Lys           310                 3 - #15                 3 - #20                 3 -       #25                                                                           - GCA CTT CAA TAT ATT GCA GAA GAG TCC CCT GA - #A TGG CCT GCC AAT AAA         1241                                                                          Ala Leu Gln Tyr Ile Ala Glu Glu Ser Pro Gl - #u Trp Pro Ala Asn Lys           #               340                                                           - TAC ATC CTT TAT AAT CCG ACA CAT TTA GAA AC - #A GAA AAT TTA TTT AAC         1289                                                                          Tyr Ile Leu Tyr Asn Pro Thr His Leu Glu Th - #r Glu Asn Leu Phe Asn           #           355                                                               - ATC ATG ATG AAG CGA ACA GAA CAG CGG ATG CT - #T GAA GAT AGT GAT GTA         1337                                                                          Ile Met Met Lys Arg Thr Glu Gln Arg Met Le - #u Glu Asp Ser Asp Val           #       370                                                                   - CAG ATT AGA TCA AAT TCA GAA GCT ACC CGT GA - #C TAT GCT CTT TCA TTA         1385                                                                          Gln Ile Arg Ser Asn Ser Glu Ala Thr Arg As - #p Tyr Ala Leu Ser Leu           #   385                                                                       - CTC GAA ACC TTT ATT TCA CAG TTA TCT GCA AT - #A GAT ATG TTA GTA CCA         1433                                                                          Leu Glu Thr Phe Ile Ser Gln Leu Ser Ala Il - #e Asp Met Leu Val Pro           390                 3 - #95                 4 - #00                 4 -       #05                                                                           - GCA GTA GGT ATC CCA ATT AAT TTT GCC CTA TC - #A GCT ACA GCA TTA GGA         1481                                                                          Ala Val Gly Ile Pro Ile Asn Phe Ala Leu Se - #r Ala Thr Ala Leu Gly           #               420                                                           - CTT AGC TCG GAT ATT GTA GTT AAT GGA GAT TC - #A TAT GAA AAG AGA AAA         1529                                                                          Leu Ser Ser Asp Ile Val Val Asn Gly Asp Se - #r Tyr Glu Lys Arg Lys           #           435                                                               - TAT GGA ATT GGG TCC TTA GTG CAA TCT GCA TT - #A TTC ACA GGA ATT AAT         1577                                                                          Tyr Gly Ile Gly Ser Leu Val Gln Ser Ala Le - #u Phe Thr Gly Ile Asn           #       450                                                                   - CTT ATT CCA GTT ATT TCG GAA ACC GCA GAA AT - #T TTA TCT TCT TTC TCT         1625                                                                          Leu Ile Pro Val Ile Ser Glu Thr Ala Glu Il - #e Leu Ser Ser Phe Ser           #   465                                                                       - AGA ACA GAA GAA GAT ATT CCA GCT TTT TTC AC - #T GAA GAA CAA GCT TTA         1673                                                                          Arg Thr Glu Glu Asp Ile Pro Ala Phe Phe Th - #r Glu Glu Gln Ala Leu           470                 4 - #75                 4 - #80                 4 -       #85                                                                           - GCT CAA CGC TTT GAA ATA GTA GAA GAA GAA TT - #A CAT TCT ATC TCA CCT         1721                                                                          Ala Gln Arg Phe Glu Ile Val Glu Glu Glu Le - #u His Ser Ile Ser Pro           #               500                                                           - GAT GAT CCT CCT CGA GAA ATT ACT GAC GAA AA - #T TTA CAT AAA ATT CGT         1769                                                                          Asp Asp Pro Pro Arg Glu Ile Thr Asp Glu As - #n Leu His Lys Ile Arg           #           515                                                               - CTG GTA CGT CTT AAC AAT GAA AAT CAA CCT TT - #A GTT GTG TTA CGA AGA         1817                                                                          Leu Val Arg Leu Asn Asn Glu Asn Gln Pro Le - #u Val Val Leu Arg Arg           #       530                                                                   - TTA GGA GGA AAT AAA TTT ATC AGA ATC GAG CC - #T ATA ACA TTC CAG GAA         1865                                                                          Leu Gly Gly Asn Lys Phe Ile Arg Ile Glu Pr - #o Ile Thr Phe Gln Glu           #   545                                                                       - ATA AAA GGT TCT TTA GTA AGT GAA GTT ATA AA - #T CCA GTG ACT AAT AAA         1913                                                                          Ile Lys Gly Ser Leu Val Ser Glu Val Ile As - #n Pro Val Thr Asn Lys           550                 5 - #55                 5 - #60                 5 -       #65                                                                           - ACG TAC TAC GTA AGC AAT GCT AAA CTA TTA GG - #G GGC TCT CCT TAT AGT         1961                                                                          Thr Tyr Tyr Val Ser Asn Ala Lys Leu Leu Gl - #y Gly Ser Pro Tyr Ser           #               580                                                           - CCT TTC CGT ATT GGA TTA GAA GGT GTT TGG AC - #A CCA GAG GTA TTA AAA         2009                                                                          Pro Phe Arg Ile Gly Leu Glu Gly Val Trp Th - #r Pro Glu Val Leu Lys           #           595                                                               - GCA AGA GCT TCC GTT ATT GGA AAG CCT ATT GG - #A GAA TCA TAT AAA AGA         2057                                                                          Ala Arg Ala Ser Val Ile Gly Lys Pro Ile Gl - #y Glu Ser Tyr Lys Arg           #       610                                                                   - ATA TTA GCC AAA CTA CAA AGA ATA CAT AAC AG - #T AAT ATC TTA GAT GAG         2105                                                                          Ile Leu Ala Lys Leu Gln Arg Ile His Asn Se - #r Asn Ile Leu Asp Glu           #   625                                                                       - CGA CAA GGT TTA ATG CAT GAA CTC ATG GAG CT - #T ATT GAT CTT TAT GAA         2153                                                                          Arg Gln Gly Leu Met His Glu Leu Met Glu Le - #u Ile Asp Leu Tyr Glu           630                 6 - #35                 6 - #40                 6 -       #45                                                                           - GAA TCG CAA CCT TCT TCA GAG CGT TTG AAT GC - #T TTT CGT GAA CTG CGT         2201                                                                          Glu Ser Gln Pro Ser Ser Glu Arg Leu Asn Al - #a Phe Arg Glu Leu Arg           #               660                                                           - ACT CAA TTA GAA AAA GCG CTT TAT CTT CCT GA - #A ATG GAA GCA TTA AAA         2249                                                                          Thr Gln Leu Glu Lys Ala Leu Tyr Leu Pro Gl - #u Met Glu Ala Leu Lys           #           675                                                               - AAA CAA ATA CTA CAG ATT CCT AAC AAA GGT TC - #T GGT GCC GCT CGA TTT         2297                                                                          Lys Gln Ile Leu Gln Ile Pro Asn Lys Gly Se - #r Gly Ala Ala Arg Phe           #       690                                                                   - TTA CTT CGT ACA GCC ATG AAT GAA ATG GCT GG - #A AAA ACC AGT GAA AGC         2345                                                                          Leu Leu Arg Thr Ala Met Asn Glu Met Ala Gl - #y Lys Thr Ser Glu Ser           #   705                                                                       - ACG GCT GAT TTA ATA CGC TTT GCC TTG CAA GA - #T ACA GTA ATT TCA GCG         2393                                                                          Thr Ala Asp Leu Ile Arg Phe Ala Leu Gln As - #p Thr Val Ile Ser Ala           710                 7 - #15                 7 - #20                 7 -       #25                                                                           - CCT TTT CGC GGA TAT GCT GGT GCG ATT CCA GA - #G GCA ATA GAC TTT CCT         2441                                                                          Pro Phe Arg Gly Tyr Ala Gly Ala Ile Pro Gl - #u Ala Ile Asp Phe Pro           #               740                                                           - GTA AAA TAT GTA ATA GAA GAC ATA TCT GTA TT - #T GAT AAA ATA CAG ACA         2489                                                                          Val Lys Tyr Val Ile Glu Asp Ile Ser Val Ph - #e Asp Lys Ile Gln Thr           #           755                                                               - AAT TAC TGG GAA CTT CCT GCT TAT GAA AGC TG - #G AAC GAA GGA AGT AAT         2537                                                                          Asn Tyr Trp Glu Leu Pro Ala Tyr Glu Ser Tr - #p Asn Glu Gly Ser Asn           #       770                                                                   - AGC CGA TTA CTG CCT GGT TTG TTA CGT GAA TC - #G CAA AGC AAG GGG ATG         2585                                                                          Ser Arg Leu Leu Pro Gly Leu Leu Arg Glu Se - #r Gln Ser Lys Gly Met           #   785                                                                       - TTA AGT AAG TGT CGT ATC ATA GAA AAT AGC CT - #T TAT ATT GGA CAT AGC         2633                                                                          Leu Ser Lys Cys Arg Ile Ile Glu Asn Ser Le - #u Tyr Ile Gly His Ser           790                 7 - #95                 8 - #00                 8 -       #05                                                                           - TAT GAA GAA ATG TTT TAC AGC ATT TCT CCA TA - #T TCA AAC CAG GTT GGA         2681                                                                          Tyr Glu Glu Met Phe Tyr Ser Ile Ser Pro Ty - #r Ser Asn Gln Val Gly           #               820                                                           - GGG CCT TAT GAA TTA TAT CCT TTC ACT TTT TT - #C AGT ATG CTT CAA GAA         2729                                                                          Gly Pro Tyr Glu Leu Tyr Pro Phe Thr Phe Ph - #e Ser Met Leu Gln Glu           #           835                                                               - GTA CAA GGT GAT TTA GGA TTT GAG CAG GCC TT - #T GCC ACA CGT AAC TTT         2777                                                                          Val Gln Gly Asp Leu Gly Phe Glu Gln Ala Ph - #e Ala Thr Arg Asn Phe           #       850                                                                   - TTC AAT ACT CTT GTT TCT GAT CGA CTA TCC TT - #A ATG GAA AAT ACG ATG         2825                                                                          Phe Asn Thr Leu Val Ser Asp Arg Leu Ser Le - #u Met Glu Asn Thr Met           #   865                                                                       - TTA CTT ACA GAA AGT TTT GAT TAT ACA CCT TG - #G GAT GCT ATT TAT GGA         2873                                                                          Leu Leu Thr Glu Ser Phe Asp Tyr Thr Pro Tr - #p Asp Ala Ile Tyr Gly           870                 8 - #75                 8 - #80                 8 -       #85                                                                           - GAT ATT AAT TAT GAT GAA CAA TTT GCT GCA AT - #G TCT ATT AAT GAA CGC         2921                                                                          Asp Ile Asn Tyr Asp Glu Gln Phe Ala Ala Me - #t Ser Ile Asn Glu Arg           #               900                                                           - ATA GAA AAA TGT ATG AAT ACC TAT AGA GGT GT - #G GCA TTC CAA AAC TCT         2969                                                                          Ile Glu Lys Cys Met Asn Thr Tyr Arg Gly Va - #l Ala Phe Gln Asn Ser           #           915                                                               - TCA AAA AGT ATT GAC TTT TTC CTA AAT AAT CT - #A ACC ACA TTC ATT GAT         3017                                                                          Ser Lys Ser Ile Asp Phe Phe Leu Asn Asn Le - #u Thr Thr Phe Ile Asp           #       930                                                                   - AAT GGA CTA ACC GAA ATT GCT ATA TCT GAT TT - #A CCG TAT GAT ATT GTG         3065                                                                          Asn Gly Leu Thr Glu Ile Ala Ile Ser Asp Le - #u Pro Tyr Asp Ile Val           #   945                                                                       - CAA CAA GAA ATC TCT CAA TTC TTA CAA GGA AG - #T AAT GAA TGG AAA ACA         3113                                                                          Gln Gln Glu Ile Ser Gln Phe Leu Gln Gly Se - #r Asn Glu Trp Lys Thr           950                 9 - #55                 9 - #60                 9 -       #65                                                                           - CTT GAT GCC ATG TTA TTT AAC TTA GAT AAA GG - #A GAT ATT AAT GGT GCT         3161                                                                          Leu Asp Ala Met Leu Phe Asn Leu Asp Lys Gl - #y Asp Ile Asn Gly Ala           #               980                                                           - TTC AGA AAG CTT CTG CAA TCA GCA AAA GAT AA - #T AAT ATA AAA TTT AGA         3209                                                                          Phe Arg Lys Leu Leu Gln Ser Ala Lys Asp As - #n Asn Ile Lys Phe Arg           #           995                                                               - GCT ATA GGG CAT TCA GAT AAT TCT GTT CCG CC - #A TTT AAT AAC CCT TAT         3257                                                                          Ala Ile Gly His Ser Asp Asn Ser Val Pro Pr - #o Phe Asn Asn Pro Tyr           #      10105                                                                  - AAG TCT TTA TAT TAT AAA GGA AAT ATA ATA GC - #T GAA GCA ATT GAA AAA         3305                                                                          Lys Ser Leu Tyr Tyr Lys Gly Asn Ile Ile Al - #a Glu Ala Ile Glu Lys           #  10250                                                                      - CTA GAT CGA GAA GGT CAA AAA TTT GTT GTA TT - #T GCT GAT AGT TCT CTG         3353                                                                          Leu Asp Arg Glu Gly Gln Lys Phe Val Val Ph - #e Ala Asp Ser Ser Leu           #               10451035 - #                1040                              - CTC AAC AGC ACG CCT GGG ACA GGT CGT CCT AT - #G CCA GGA CTA GTT CAA         3401                                                                          Leu Asn Ser Thr Pro Gly Thr Gly Arg Pro Me - #t Pro Gly Leu Val Gln           #              10605                                                          - TAT TTA AAA ATA CCA GCA ACT GTA GTA GAT AG - #C GAT GGT GCA TGG CAA         3449                                                                          Tyr Leu Lys Ile Pro Ala Thr Val Val Asp Se - #r Asp Gly Ala Trp Gln           #          10750                                                              - TTT CTT CCA GAT GTA GCT TCA AGC AGA GTT CC - #T ATT GAA GTT ACA GAG         3497                                                                          Phe Leu Pro Asp Val Ala Ser Ser Arg Val Pr - #o Ile Glu Val Thr Glu           #      10905                                                                  - TTA GAA AAT TGG CAA GTC TTA ACT CCT CCA CA - #A GGT AAG ATT CTT GGA         3545                                                                          Leu Glu Asn Trp Gln Val Leu Thr Pro Pro Gl - #n Gly Lys Ile Leu Gly           #  11050                                                                      - TTA AAG CAA TTT AAG TTA ACG GCA GGT TTT CC - #A ACA GAA CAA AGT CGC         3593                                                                          Leu Lys Gln Phe Lys Leu Thr Ala Gly Phe Pr - #o Thr Glu Gln Ser Arg           #               11251115 - #                1120                              - TTA CCT CTT TTA GAG AAT TCG GTT TCT GAA GA - #T TTA AGG GAA GAA TTA         3641                                                                          Leu Pro Leu Leu Glu Asn Ser Val Ser Glu As - #p Leu Arg Glu Glu Leu           #              11405                                                          - ATG CAA AAG ATT GAT GCA ATA AAA AAT GAT GT - #G AAA ATG AAT AGT TTA         3689                                                                          Met Gln Lys Ile Asp Ala Ile Lys Asn Asp Va - #l Lys Met Asn Ser Leu           #          11550                                                              - GTG TGT ATG GAA GCT GGC TCT TGT GAT TCA GT - #A AGC CCT AAG GTA GCT         3737                                                                          Val Cys Met Glu Ala Gly Ser Cys Asp Ser Va - #l Ser Pro Lys Val Ala           #      11705                                                                  - GCC CGT CTT AAA GAT ATG GGG TTA GAA GCT GG - #G ATG GGT GCT TCT ATT         3785                                                                          Ala Arg Leu Lys Asp Met Gly Leu Glu Ala Gl - #y Met Gly Ala Ser Ile           #  11850                                                                      - ACC TGG TGG AGA CGT GAA GGC GGG ATG GAA TT - #T TCA CAT CAG ATG CAT         3833                                                                          Thr Trp Trp Arg Arg Glu Gly Gly Met Glu Ph - #e Ser His Gln Met His           #               12051195 - #                1200                              - ACT ACT GCT TCC TTT AAA TTT GCT GGT AAA GA - #G TTT GCC GTG GAT GCT         3881                                                                          Thr Thr Ala Ser Phe Lys Phe Ala Gly Lys Gl - #u Phe Ala Val Asp Ala           #              12205                                                          - TCA CAT TTA CAA TTT GTA CAC GAC CAA TTA GA - #T ACA ACT ATC CTG ATA         3929                                                                          Ser His Leu Gln Phe Val His Asp Gln Leu As - #p Thr Thr Ile Leu Ile           #          12350                                                              - CTA CCT GTA GAT GAT TGG GCT TTA GAA ATA GC - #T CAA AGA AAT CGG GCT         3977                                                                          Leu Pro Val Asp Asp Trp Ala Leu Glu Ile Al - #a Gln Arg Asn Arg Ala           #      12505                                                                  - ATT AAT CCT TTT GTG GAA TAT GTT AGT AAA AC - #A GGA AAC ATG TTA GCA         4025                                                                          Ile Asn Pro Phe Val Glu Tyr Val Ser Lys Th - #r Gly Asn Met Leu Ala           #  12650                                                                      - CTC TTC ATG CCT CCT CTT TTC ACA AAG CCT CG - #C TTA ACA AGA GCA CTA         4073                                                                          Leu Phe Met Pro Pro Leu Phe Thr Lys Pro Ar - #g Leu Thr Arg Ala Leu           #               12851275 - #                1280                              - TAACTAATTA AAAACTGTAT TAAAGCCTTA TATTATAAGG CTTTAATTTT CT - #TTCAAGAA       4133                                                                          - TTATTAAGTA GAAGAATCAA AATCAATGAG ATAGATAAAA TCAAATGTTA TT - #ACCAATAC       4193                                                                          - AACTTTCTTA AGTATACTTT TTGAATTTTT TGCGTTAATA AATTTATAAT AC - #CCTTAACT       4253                                                                          - CAATAAAAGA AGTTATTGAG AAGTTTAAAT CTTGTGAGCA AGATGAAGAT AT - #AATTTCAG       4313                                                                          - CAATCGATCT TATTAGCGCT TCATATAGAA GGGCTGTGGA TGCAGTGGAA CA - #AAGATTCG       4373                                                                          #        4380                                                                 - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1285 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Lys Thr Lys His Phe Phe Asn Ser Asp Ph - #e Thr Val Lys Gly Lys         #                 15                                                          - Ser Ala Asp Glu Ile Phe Arg Arg Leu Cys Th - #r Asp His Pro Asp Lys         #             30                                                              - Gln Leu Asn Asn Val Lys Trp Lys Glu Val Ph - #e Ile Asn Arg Phe Gly         #         45                                                                  - Gln Met Met Leu Asp Thr Pro Asn Pro Arg Ly - #s Ile Val Glu Lys Ile         #     60                                                                      - Ile Asn Glu Gly Leu Glu Lys Gln Gly Leu Ly - #s Asn Ile Asp Pro Glu         # 80                                                                          - Thr Thr Tyr Phe Asn Ile Phe Ser Ser Ser As - #p Ser Ser Asp Gly Asn         #                 95                                                          - Val Phe His Tyr Asn Ser Leu Ser Glu Ser Ty - #r Arg Val Thr Asp Ala         #           110                                                               - Cys Leu Met Asn Ile Phe Val Glu Arg Tyr Ph - #e Asp Asp Trp Asp Leu         #       125                                                                   - Leu Asn Ser Leu Ala Ser Asn Gly Ile Tyr Se - #r Val Gly Lys Glu Gly         #   140                                                                       - Ala Tyr Tyr Pro Asp His Asp Tyr Gly Pro Gl - #u Tyr Asn Pro Val Trp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Pro Asn Glu Gln Ile Tyr His Ser Arg Va - #l Ile Ala Asp Ile Leu         #               175                                                           - Tyr Ala Arg Ser Val Trp Asp Glu Phe Lys Ly - #s Tyr Phe Met Glu Tyr         #           190                                                               - Trp Gln Lys Tyr Ala Gln Leu Tyr Thr Glu Me - #t Leu Ser Asp Thr Phe         #       205                                                                   - Leu Ala Met Ala Ile Gln Gln Tyr Thr Arg Gl - #n Thr Leu Thr Asp Glu         #   220                                                                       - Gly Phe Leu Met Val Cys Asn Thr Tyr Tyr Gl - #y Asn Lys Glu Glu Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gln Ile Thr Leu Leu Asp Ile Tyr Gly Tyr Pr - #o Ser Thr Asp Ile Ile         #               255                                                           - Cys Ile Glu Gln Lys Gly Leu Pro Thr Pro Ly - #s Val Ile Leu Tyr Ile         #           270                                                               - Pro Gly Gly Thr Gln Pro Phe Val Glu Phe Le - #u Asn Thr Asp Asp Leu         #       285                                                                   - Lys Gln Trp Ile Ala Trp His Leu Lys Asp As - #n Lys His Met Val Arg         #   300                                                                       - Phe Arg Lys His Phe Ser Leu Lys Gln Arg Gl - #n Glu Gly Glu Thr Phe         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Thr Gly Ile Asp Lys Ala Leu Gln Tyr Ile Al - #a Glu Glu Ser Pro Glu         #               335                                                           - Trp Pro Ala Asn Lys Tyr Ile Leu Tyr Asn Pr - #o Thr His Leu Glu Thr         #           350                                                               - Glu Asn Leu Phe Asn Ile Met Met Lys Arg Th - #r Glu Gln Arg Met Leu         #       365                                                                   - Glu Asp Ser Asp Val Gln Ile Arg Ser Asn Se - #r Glu Ala Thr Arg Asp         #   380                                                                       - Tyr Ala Leu Ser Leu Leu Glu Thr Phe Ile Se - #r Gln Leu Ser Ala Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asp Met Leu Val Pro Ala Val Gly Ile Pro Il - #e Asn Phe Ala Leu Ser         #               415                                                           - Ala Thr Ala Leu Gly Leu Ser Ser Asp Ile Va - #l Val Asn Gly Asp Ser         #           430                                                               - Tyr Glu Lys Arg Lys Tyr Gly Ile Gly Ser Le - #u Val Gln Ser Ala Leu         #       445                                                                   - Phe Thr Gly Ile Asn Leu Ile Pro Val Ile Se - #r Glu Thr Ala Glu Ile         #   460                                                                       - Leu Ser Ser Phe Ser Arg Thr Glu Glu Asp Il - #e Pro Ala Phe Phe Thr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Glu Glu Gln Ala Leu Ala Gln Arg Phe Glu Il - #e Val Glu Glu Glu Leu         #               495                                                           - His Ser Ile Ser Pro Asp Asp Pro Pro Arg Gl - #u Ile Thr Asp Glu Asn         #           510                                                               - Leu His Lys Ile Arg Leu Val Arg Leu Asn As - #n Glu Asn Gln Pro Leu         #       525                                                                   - Val Val Leu Arg Arg Leu Gly Gly Asn Lys Ph - #e Ile Arg Ile Glu Pro         #   540                                                                       - Ile Thr Phe Gln Glu Ile Lys Gly Ser Leu Va - #l Ser Glu Val Ile Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Pro Val Thr Asn Lys Thr Tyr Tyr Val Ser As - #n Ala Lys Leu Leu Gly         #               575                                                           - Gly Ser Pro Tyr Ser Pro Phe Arg Ile Gly Le - #u Glu Gly Val Trp Thr         #           590                                                               - Pro Glu Val Leu Lys Ala Arg Ala Ser Val Il - #e Gly Lys Pro Ile Gly         #       605                                                                   - Glu Ser Tyr Lys Arg Ile Leu Ala Lys Leu Gl - #n Arg Ile His Asn Ser         #   620                                                                       - Asn Ile Leu Asp Glu Arg Gln Gly Leu Met Hi - #s Glu Leu Met Glu Leu         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ile Asp Leu Tyr Glu Glu Ser Gln Pro Ser Se - #r Glu Arg Leu Asn Ala         #               655                                                           - Phe Arg Glu Leu Arg Thr Gln Leu Glu Lys Al - #a Leu Tyr Leu Pro Glu         #           670                                                               - Met Glu Ala Leu Lys Lys Gln Ile Leu Gln Il - #e Pro Asn Lys Gly Ser         #       685                                                                   - Gly Ala Ala Arg Phe Leu Leu Arg Thr Ala Me - #t Asn Glu Met Ala Gly         #   700                                                                       - Lys Thr Ser Glu Ser Thr Ala Asp Leu Ile Ar - #g Phe Ala Leu Gln Asp         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Thr Val Ile Ser Ala Pro Phe Arg Gly Tyr Al - #a Gly Ala Ile Pro Glu         #               735                                                           - Ala Ile Asp Phe Pro Val Lys Tyr Val Ile Gl - #u Asp Ile Ser Val Phe         #           750                                                               - Asp Lys Ile Gln Thr Asn Tyr Trp Glu Leu Pr - #o Ala Tyr Glu Ser Trp         #       765                                                                   - Asn Glu Gly Ser Asn Ser Arg Leu Leu Pro Gl - #y Leu Leu Arg Glu Ser         #   780                                                                       - Gln Ser Lys Gly Met Leu Ser Lys Cys Arg Il - #e Ile Glu Asn Ser Leu         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Tyr Ile Gly His Ser Tyr Glu Glu Met Phe Ty - #r Ser Ile Ser Pro Tyr         #               815                                                           - Ser Asn Gln Val Gly Gly Pro Tyr Glu Leu Ty - #r Pro Phe Thr Phe Phe         #           830                                                               - Ser Met Leu Gln Glu Val Gln Gly Asp Leu Gl - #y Phe Glu Gln Ala Phe         #       845                                                                   - Ala Thr Arg Asn Phe Phe Asn Thr Leu Val Se - #r Asp Arg Leu Ser Leu         #   860                                                                       - Met Glu Asn Thr Met Leu Leu Thr Glu Ser Ph - #e Asp Tyr Thr Pro Trp         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Asp Ala Ile Tyr Gly Asp Ile Asn Tyr Asp Gl - #u Gln Phe Ala Ala Met         #               895                                                           - Ser Ile Asn Glu Arg Ile Glu Lys Cys Met As - #n Thr Tyr Arg Gly Val         #           910                                                               - Ala Phe Gln Asn Ser Ser Lys Ser Ile Asp Ph - #e Phe Leu Asn Asn Leu         #       925                                                                   - Thr Thr Phe Ile Asp Asn Gly Leu Thr Glu Il - #e Ala Ile Ser Asp Leu         #   940                                                                       - Pro Tyr Asp Ile Val Gln Gln Glu Ile Ser Gl - #n Phe Leu Gln Gly Ser         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Asn Glu Trp Lys Thr Leu Asp Ala Met Leu Ph - #e Asn Leu Asp Lys Gly         #               975                                                           - Asp Ile Asn Gly Ala Phe Arg Lys Leu Leu Gl - #n Ser Ala Lys Asp Asn         #           990                                                               - Asn Ile Lys Phe Arg Ala Ile Gly His Ser As - #p Asn Ser Val Pro Pro         #      10050                                                                  - Phe Asn Asn Pro Tyr Lys Ser Leu Tyr Tyr Ly - #s Gly Asn Ile Ile Ala         #  10205                                                                      - Glu Ala Ile Glu Lys Leu Asp Arg Glu Gly Gl - #n Lys Phe Val Val Phe         #               10401030 - #                1035                              - Ala Asp Ser Ser Leu Leu Asn Ser Thr Pro Gl - #y Thr Gly Arg Pro Met         #              10550                                                          - Pro Gly Leu Val Gln Tyr Leu Lys Ile Pro Al - #a Thr Val Val Asp Ser         #          10705                                                              - Asp Gly Ala Trp Gln Phe Leu Pro Asp Val Al - #a Ser Ser Arg Val Pro         #      10850                                                                  - Ile Glu Val Thr Glu Leu Glu Asn Trp Gln Va - #l Leu Thr Pro Pro Gln         #  11005                                                                      - Gly Lys Ile Leu Gly Leu Lys Gln Phe Lys Le - #u Thr Ala Gly Phe Pro         #               11201110 - #                1115                              - Thr Glu Gln Ser Arg Leu Pro Leu Leu Glu As - #n Ser Val Ser Glu Asp         #              11350                                                          - Leu Arg Glu Glu Leu Met Gln Lys Ile Asp Al - #a Ile Lys Asn Asp Val         #          11505                                                              - Lys Met Asn Ser Leu Val Cys Met Glu Ala Gl - #y Ser Cys Asp Ser Val         #      11650                                                                  - Ser Pro Lys Val Ala Ala Arg Leu Lys Asp Me - #t Gly Leu Glu Ala Gly         #  11805                                                                      - Met Gly Ala Ser Ile Thr Trp Trp Arg Arg Gl - #u Gly Gly Met Glu Phe         #               12001190 - #                1195                              - Ser His Gln Met His Thr Thr Ala Ser Phe Ly - #s Phe Ala Gly Lys Glu         #              12150                                                          - Phe Ala Val Asp Ala Ser His Leu Gln Phe Va - #l His Asp Gln Leu Asp         #          12305                                                              - Thr Thr Ile Leu Ile Leu Pro Val Asp Asp Tr - #p Ala Leu Glu Ile Ala         #      12450                                                                  - Gln Arg Asn Arg Ala Ile Asn Pro Phe Val Gl - #u Tyr Val Ser Lys Thr         #  12605                                                                      - Gly Asn Met Leu Ala Leu Phe Met Pro Pro Le - #u Phe Thr Lys Pro Arg         #               12801270 - #                1275                              - Leu Thr Arg Ala Leu                                                                         1285                                                          __________________________________________________________________________

What is claimed is:
 1. An isolated and purified DNA molecule selectedfrom the group consisting of:a) a DNA molecule that encodes apolypeptide derivative comprising the amino acid sequence of SEQ IDNO:1, lacking amino acids 1043-1130; b) a DNA molecule that encodes apolypeptide derivative comprising the amino acid sequence of SEQ IDNO:1, lacking amino acids 1130-1285; and c) a DNA molecule encoding adetoxified polypeptide derivative of Pasteurella multocida toxin,wherein the derivative is a truncated form of the toxin and comprises anamino acid sequence that is identical to but shorter than the amino acidsequence of the Pasteurella multocida toxin of SEQ ID NO:1, and whereinthe toxin derivative specifically binds an antibody that specificallybinds Pasteurella multocida toxin (PMT).
 2. The DNA molecule of claim 1,wherein the antibody is a monoclonal antibody.
 3. An isolated andpurified DNA molecule selected from the group consisting of a DNAmolecule that encodes a polypeptide derivative of Pasteurella multocidatoxin comprising an amino acid sequence that is identical to but shorterthan the amino acid sequence of SEQ ID NO:1, lacking amino acids1043-1130, and a DNA molecule that encodes a detoxified polypeptidederivative of Pasteurella multocida toxin comprising an amino acidsequence that is identical to but shorter than the amino acid sequenceof SEQ ID NO:1, lacking amino acids 1130-1285, and wherein thepolypeptide derivative specifically binds an antibody that specificallybinds Pasteurella multocida toxin (PMT).
 4. The DNA molecule of claim 3,wherein the antibody is a monoclonal antibody.
 5. The DNA molecule ofclaim 1, wherein the DNA molecule encodes a polypeptide derivativecomprising the amino acid sequence of SEQ ID NO:1, lacking amino acids1043-1130.
 6. The DNA molecule of claim 1, wherein the DNA moleculeencodes a polypeptide derivative comprising the amino acid sequence ofSEQ ID NO:1, lacking amino acids 1130-1285.
 7. The DNA molecule of claim1, further comprising a nucleotide sequence that encodes anotherpolypeptide, wherein the DNA molecule encodes a fusion protein.
 8. Anexpression vector comprising the DNA molecule of claim
 1. 9. Amicroorganism containing the expression vector of claim
 8. 10. Themicroorganism of claim 9, wherein the microorganism is a bacterium. 11.The microorganism of claim 10, wherein the bacterium is Escherichiacoli.
 12. A method for the production of a Pasteurella multocida toxinderivative, comprising the steps:a) transforming a suitable hostmicroorganism with a DNA molecule according to claim 1; b) culturingsaid host microorganism to express a polypeptide derivative; c)separating said polypeptide derivative from said host microorganism; andd) recovering said polypeptide derivative.
 13. The method of claim 12,further comprising the step of testing said recovered polypeptidederivative for toxicity.